Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof

ABSTRACT

The invention is related to multivalent immunogenic compositions comprising more than one S. pneumoniae polysaccharide protein conjugates, wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, wherein the serotypes of S. pneumoniae are as defined herein. In some embodiments, at least one of the polysaccharide protein conjugates is formed by a conjugation reaction comprising an aprotic solvent. In further embodiments, each of the polysaccharide protein conjugates is formed by a conjugation reaction comprising an aprotic solvent. Also provided are methods for inducing a protective immune response in a human patient comprising administering the multivalent immunogenic compositions of the invention to the patient. The multivalent immunogenic compositions are useful for providing protection against S. pneumoniae infection and diseases caused by S. pneumoniae. The compositions of the invention are also useful as part of treatment regimes that provide complementary protection for patients that have been vaccinated with a multivalent vaccine indicated for the prevention of pneumococcal disease.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of Ser. No.16/208,778 filed Dec. 4, 2018, which claims the benefit of U.S.Provisional Application No. 62/595,388, filed Dec. 6, 2017, herebyincorporated by reference in its entirety.

FIELD OF INVENTION

The present invention provides multivalent immunogenic compositionshaving distinct polysaccharide-protein conjugates. Each conjugateconsists of a capsular polysaccharide prepared from a different serotypeof Streptococcus pneumoniae conjugated to a carrier protein, preferablyCRM197. The immunogenic compositions provide broad coverage againstpneumococcal disease.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae is a Gram-positive bacterium and the mostcommon cause of invasive bacterial disease (such as pneumonia,bacteraemia, meningitis and Otitis media) in infants and young children.Pneumococcus is encapsulated with a chemically linked polysaccharidewhich confers serotype specificity. There are over 90 known serotypes ofpneumococci, and the capsule is the principle virulence determinant forpneumococci, as the capsule not only protects the inner surface of thebacteria from complement, but is itself poorly immunogenic.Polysaccharides are T-cell independent antigens, and, in most cases, cannot be processed or presented on MHC molecules to interact with T-cells.They can however, stimulate the immune system through an alternatemechanism which involves cross-linking of surface receptors on B cells.

The multivalent pneumococcal polysaccharide vaccines that have beenlicensed for many years have proved valuable in preventing pneumococcaldisease in adults, particularly, the elderly and those at high-risk.However, infants and young children respond poorly to unconjugatedpneumococcal polysaccharides. The pneumococcal conjugate vaccine,Prevnar®, containing the 7 most frequently isolated serotypes (4, 6B,9V, 14, 18C, 19F and 23F) causing invasive pneumococcal disease in youngchildren and infants at the time, was first licensed in the UnitedStates in February 2000. Following universal use of Prevnar® in theUnited States, there has been a significant reduction in invasivepneumococcal disease in children due to the serotypes present inPrevnar®. See Centers for Disease Control and Prevention, MMWR MorbMortal Wkly Rep 2005, 54(36):893-7. However, there are limitations inserotype coverage with Prevnar® in certain regions of the world and someevidence of certain emerging serotypes in the United States (forexample, 19A and others). See O'Brien et al., 2004, Am J Epidemiol159:634-44; Whitney et al., 2003, N Engl J Med 348:1737-46; Kyaw et al.,2006, N Engl J Med 354:1455-63; Hicks et al., 2007, J Infect Dis196:1346-54; Traore et al., 2009, Clin Infect Dis 48:S181-S189.

U.S. Patent Application Publication No. US 2006/0228380 describes a13-valent pneumococcal polysaccharide-protein conjugate vaccineincluding serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and23F. Chinese Patent Application Publication No. CN 101590224 A describesa 14-valent pneumococcal polysaccharide-protein conjugate vaccineincluding serotypes 1, 2, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19Fand 23F.

Other PCVs have covered 7, 10, 11, or 13 of the serotypes contained inPCV-15 (U.S. Pub. No. 2011/0195086), but immune interference has beenobserved for some serotypes (e.g. lower protection for serotype 3 inGSK's PCV-11) and lower response rates to serotype 6B in Pfizer's PCV-13(PREVNAR® 13). See Prymula et al., 2006, Lancet 367:740-48 and Kieningeret al., Safety and Immunologic Non-inferiority of 13-valent PneumococcalConjugate Vaccine Compared to 7-valent Pneumococcal Conjugate VaccineGiven as a 4-Dose Series in Healthy Infants and Toddlers, presented atthe 48^(th) Annual ICAAC/ISDA 46^(th) Annual Meeting, Washington DC,Oct. 25-28, 2008.

The current multivalent pneumococcal vaccines have been effective inreducing the incidence of pneumococcal disease associated with thoseserotypes present in the vaccines. However, the prevalence of thepneumococci expressing serotypes not present in the currently availablevaccines has been increasing. Accordingly, there is a need foradditional pneumococcal vaccine compositions which provide protectionagainst different sets of pneumococcal serotypes and which can providecomplementary protection against pneumococcal serotypes not present incurrently available vaccines.

SUMMARY OF THE INVENTION

The invention provides multivalent immunogenic compositions comprisingS. pneumoniae polysaccharide protein conjugates wherein each of theconjugates comprises a polysaccharide from an S. pneumoniae serotypeconjugated to a carrier protein, wherein the serotypes of S. pneumoniaeare as defined herein.

In particular embodiments of the invention, the serotypes of S.pneumoniae comprise a set of serotypes selected from the groupconsisting of:

-   -   a) 15A, 16F, 23A, 23B, 24F, 31 and 35B;    -   b) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C,        17F, and 20; and    -   c) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8,        9N, 10A, 11A, 12F, 15C, 17F, and 20.

In further particular embodiments of the invention, the serotypes of S.pneumoniae comprise a set of serotypes selected from the groupconsisting of:

-   -   a) 15A, 16F, 23A, 23B, 24F, 31 and 35B;    -   b) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C,        17F, and 20A; and    -   c) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8,        9N, 10A, 11A, 12F, 15C, 17F, and 20A.

In further particular embodiments of the invention, the serotypes of S.pneumoniae comprise a set of serotypes selected from the groupconsisting of:

-   -   a) 15A, 16F, 23A, 23B, 24F, 31 and 35B;    -   b) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C,        17F, and 20B; and    -   c) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8,        9N, 10A, 11A, 12F, 15C, 17F, and 20B.

In some embodiments, the set of serotypes of S. pneumoniae listed in a),b) or c) further comprises: serotype 6C, serotype 6A, or serotypes 6Aand 6B.

In some embodiments, at least one of the polysaccharide proteinconjugates is formed by a conjugation reaction comprising an aproticsolvent, e.g. dimethylsulfoxide (DMSO).

In specific embodiments, each of the polysaccharide protein conjugatesis formed by a conjugation reaction comprising an aprotic solvent. Asshown herein, the use of DMSO as a solvent during reductive amination ofpolysaccharide-protein conjugates results in unexpectedly superiorstability and enhanced immunogenicity for those serotypes relative tothe same conjugates prepared under aqueous conditions.

Also provided are methods for inducing a protective immune response in ahuman patient comprising administering the multivalent immunogeniccompositions of the invention to the patient. In some embodiments of themethods of the invention, the patient was previously treated with amultivalent pneumococcal vaccine.

A multivalent immunogenic composition of the invention may be used aspart of a treatment regimen with a different, complementary pneumococcalvaccine. Accordingly, the invention provides a method of inducing aprotective immune response in a human patient comprising administering amultivalent immunogenic composition of the invention to the patient,further comprising administering a multivalent pneumococcal vaccine tothe patient in any order. In particular embodiments, the multivalentpneumococcal vaccine is comprised of multiple S. pneumoniaepolysaccharide protein conjugates wherein each of the conjugatescomprises polysaccharide from an S. pneumoniae serotype conjugated to acarrier protein. In other embodiments, the multivalent pneumococcalvaccine is comprised of unconjugated capsular polysaccharides.

Also provided are methods for preparing a serotype 8 Streptococcuspneumoniae polysaccharide-protein conjugate utilizing a conjugationreaction in an aprotic solvent, wherein the conjugation reaction doesnot use cyanoborohydride.

The invention also provides multivalent immunogenic compositionscomprising S. pneumoniae polysaccharide protein conjugates wherein eachof the conjugates comprises a polysaccharide from an S. pneumoniaeserotype conjugated to a carrier protein, wherein select serotypes of S.pneumoniae provide cross-reactivity to other select serotypes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the 600 MHz one-dimensional ¹H NMR spectrum of thecapsular polysaccharide from S. pneumonia serotype 6C in deuterium oxide(D₂O) at 50° C. Signals arising from internal standards (DMSO andDSS-d₆) and residual water (HOD) are marked. Minor signals marked by *are due to S. pneumonia cell wall residuals such as C-polysaccharideand/or peptidoglycans.

FIG. 2 depicts the 600 MHz one-dimensional ¹H NMR spectrum of thecapsular polysaccharide from S. pneumonia serotype 15A in deuteriumoxide (D₂O) at 50° C. Signals arising from internal standards (DMSO andDSS-d₆) and residual water (HOD) are marked. Minor signals marked by *are due to S. pneumonia cell wall residuals such as C-polysaccharideand/or peptidoglycans.

FIG. 3 depicts the 600 MHz one-dimensional ¹H NMR spectrum of thecapsular polysaccharide from S. pneumonia serotype de-O-acetylated 15Bin deuterium oxide (D₂O) at 50° C. Signals arising from internalstandards (DMSO and DSS-d₆) and residual water (HOD) are marked. Minorsignals marked by * are due to S. pneumonia cell wall residuals such asC-polysaccharide and/or peptidoglycans.

FIG. 4 depicts the 600 MHz one-dimensional ¹H NMR spectrum of thecapsular polysaccharide from S. pneumonia serotype 35B in D₂O at 50° C.Signals arising from internal standards (DMSO and DSS-d₆) and residualwater (HOD) are marked. Minor signals marked by * are due to S.pneumonia cell wall residuals such as C-polysaccharide and/orpeptidoglycans.

FIG. 5 depicts the ¹H NMR identity region useful for serotypeidentification of S. pneumonia serotype 6C. Signal positions of eachanomeric proton of the repeating unit from each monosaccharide residueare marked.

FIG. 6 depicts the ¹H NMR identity region useful for serotypeidentification of S. pneumonia serotype 15A. Signal positions of eachanomeric proton of the repeating unit from each monosaccharide residueare marked.

FIG. 7 depicts the ¹H NMR identity region useful for serotypeidentification of S. pneumonia serotype de-O-acetylated 15B. Signalpositions of each anomeric proton from each monosaccharide residue aremarked.

FIG. 8 depicts the ¹H NMR identity region useful for serotypeidentification of S. pneumonia serotype 35B. Signal positions of eachanomeric proton of the repeating unit from each monosaccharide residueare marked.

FIGS. 9A-9C depict the 600 MHz one-dimensional ¹H NMR spectrum of nativecapsular polysaccharide from S. pneumonia serotype 15B (FIG. 9A), thede-O-acetylated capsular polysaccharide from S. pneumonia serotype 15B(FIG. 9B) and the capsular polysaccharide from S. pneumonia serotype 15C(FIG. 9C). Spectra were acquired in deuterium oxide (D₂O) at 50° C.Signals arising from internal standards (DMSO and DSS-d₆) and residualwater (HOD) are marked. Signals marked by * are due to S. pneumonia cellwall residuals such as C-polysaccharide and/or peptidoglycans.

FIGS. 10A-10C show the anomeric region of the 600 MHz one-dimensional ¹HNMR spectrum of native capsular polysaccharide from S. pneumoniaserotype 15B (FIG. 10A), the de-O-acetylated capsular polysaccharidefrom S. pneumonia serotype 15B (FIG. 10B) and the capsularpolysaccharide from S. pneumonia serotype 15C (FIG. 10C). FIGS. 10D-10Fshow regions of the spectrum where the O-acetyl and N-acetyl methysignals of native capsular polysaccharide from S. pneumonia serotype 15B(FIG. 10D) the de-O-acetylated capsular polysaccharide from S. pneumoniaserotype 15B (FIG. 10E) and the capsular polysaccharide from S.pneumonia serotype 15C (FIG. 10F).

FIG. 11 shows the impact of time and temperature (up to 12 weeks at 4°C.-diamonds, up to 4 weeks at 25° C.-squares, up to 4 weeks at 37°C.-triangles) on the polysaccharide concentration of a PCV16 (0.128mg/mL) or PCV21 (0.084 mg/mL or 0.169 mg/mL) drug products usingHPSEC-UV/MALS/RI (see EXAMPLE 39).

FIG. 12 shows the impact of horizontal rotation agitation andtemperature (1 week at either 4° C., 25° C., or 37° C.) onpolysaccharide concentration of a PCV16 (0.128 mg/mL) or PCV21 (0.084mg/mL or 0.169 mg/mL) drug products dispensed in pre-filled syringes(see EXAMPLE 39).

FIG. 13 shows the impact of PS concentration over time and temperature(up to 4 weeks at either 4° C., 25° C., or 37° C.) on the averagemolecular weight (Mw and Mn) of PCV16 (0.128 mg/mL) or PCV21 (0.084mg/mL or 0.169 mg/mL) drug products using HPSEC-UV/MALS/RI.

FIGS. 14A and 14B show the impact of time and temperature (up to 1 wk at4° C. or 37° C.) on stability of a Pneumococcal Conjugate vaccine (PCV15or PCV16) prepared with drug substances conjugated in A) a proticsolvent (PCV15 formulated using all aqueous conjugation) or B) anaprotic solvent (PCV16 formulated using all DMSO conjugation at 0.064mg/mL PnPs) using intrinsic protein fluorescence spectroscopy with anexcitation wavelength at 280 nm (EXAMPLE 40).

FIG. 15 shows ELISA IgG antibody dilution titers (post-dose 2) forrabbits immunized with S. pneumoniae monovalent serotypes conjugated toCRM197 and formulated with aluminum phosphate adjuvant (APA). Symbolsindicate the individual titers and error bars represent the 95%confidence intervals (CIs) of the geometric mean titers (GMTs).

FIG. 16 provides serotype specific OPA dilution titers (post-dose 2) forrabbits immunized with S. pneumoniae monovalent serotypes conjugated toCRM197 and formulated with aluminum phosphate adjuvant (APA). Symbolsindicate the individual titers and error bars represent the 95%confidence intervals (CIs) of the geometric mean titers (GMTs).

FIG. 17 provides ELISA IgG antibody dilution titers (post-dose 2) forrabbits immunized with S. pneumoniae serotype 15 monovalent conjugates.X-axis indicates the vaccine used to immunize rabbits. Dashed linesseparate three separate ELISA assays using separate pneumococcalpolysaccharide as a coating antigen. Symbols indicate the individualtiters and error bars represent the 95% confidence intervals (CIs) ofthe geometric mean titers (GMTs).

FIG. 18 shows serotype specific OPA dilution titers (pre-immune,post-dose 1 (PD1, pooled) and post-dose 2 (PD2)) for rabbits immunizedwith S. pneumoniae serotype 15 monovalent conjugates. X-axis indicatesthe vaccine used to immunize rabbits. Dashed lines separate three OPAassays using separate S. pneumoniae bacterial strains. Symbols indicatethe individual titers and error bars represent the 95% confidenceintervals (CIs) of the geometric mean titers (GMTs).

FIG. 19A provides a comparison of PD1 antibody responses in NZWR (5 pergroup) following vaccination with 4, 2, 1, 0.4, 0.08 or 0.016 μg/dose ofPCV21. Symbols indicate PD1 geometric mean titer (GMT) ratios (Group 2μg/dose vs. other dose groups) with error bars representing the 95%confidence interval (CI). FIG. 19B provides a PCV21 dose comparison PD1GMT ratios (95% CI) corresponding to FIG. 19A GMT ratios whose lower 95%confidence bound exceed 1.0 are shaded light gray and GMT ratios whoseupper 95% confidence bound are less than 1.0 are shaded dark gray.Serotype 15B data is included to evaluate cross protection.

FIG. 20A provides a comparison of PD2 antibody responses in NZWR (5 pergroup) following vaccination with 4, 2, 1, 0.4, 0.08 or 0.016 μg/dose ofPCV21. Symbols indicate PD2 GMT ratios (Group 2 μg/dose vs. other dosegroups) with error bars representing the 95% CI. FIG. 20B provides aPCV21 dose comparison PD2 GMT ratios (95% confidence interval)corresponding to FIG. 20A GMT ratios whose lower 95% confidence boundexceed 1.0 are shaded light gray. Serotype 15B data is included toevaluate cross protection.

FIG. 21A shows the serotype specific PD1 OPA dilution titers for rabbitsimmunized with PCV21 (2 μg/PnPs). Symbols indicate the individual titersand error bars represent the 95% confidence intervals (CIs) of thegeometric mean titers (GMTs).*p<0.05, ** p<0.01, ***p<0.001,****p<0.0001. FIG. 21B shows serotype specific PD2 OPA dilution titersfor rabbits immunized with PCV21 (2 μg/PnPs). Symbols indicate theindividual titers and error bars represent the 95% confidence intervals(CIs) of the geometric mean titers (GMTs), ****p<0.0001. Serotype 15Bdata is included to evaluate cross protection.

FIG. 22A-C show the impact of time and temperature (i.e. 7 Days at 4°C., 1 Day at 37° C. or 7 Days at 37° C.) on stability of threePneumococcal Conjugate vaccines (PCV1, PCV7, and PCV14), prepared withall drug substances conjugated in either aqueous solvent or DMSOsolvent, using intrinsic protein fluorescence spectroscopy with anexcitation wavelength at 280 nm. FIG. 22D shows the impact of time andtemperature (i.e. 7 Days at 4° C. or 37° C.) on stability of a 21-valentPneumococcal Conjugate vaccine (PCV21 at 0.084 mg/mL PnPs), preparedwith all drug substances conjugated in DMSO solvent, using intrinsicprotein fluorescence spectroscopy with an excitation wavelength at 280nm.

FIG. 23 shows the impact of temperature (7 Days at 4° C. or 37° C.) andagitation at 4° C. on the particle size distribution as analyzed byNanoparticle Tracking Analysis (NTA) of six (6) PCV21 PneumococcalConjugate vaccine drug products formulated with PS-20 (0%, 0.025%,0.05%, 0.1%, 0.15% or 0.2%; all w/v PS-20) at 0.084 mg/mL PnPs.

FIG. 24 shows the impact of temperature (4° C. and 37° C.) and agitationon the three PCV21 drug product average molecular weight as analyzed byHPSEC/UV/MALS/RI assay. Three PCV21 Pneumococcal Conjugate vaccine drugproducts were formulated with different concentrations of PS-20 (0.05%,0.1%, and 0.15% w/v) at 0.084 mg/mL PnPs.

FIG. 25 shows that PCV21 immunized mice are protected from S. pneumoniae24F intratracheal challenge.

FIG. 26A-B shows pre (pooled), PD1 (pooled) and PD2 IgG antibodydilution titers as determined by ECL for rabbits immunized with PCV21unadjuvanted or formulated with APA. Error bars represent the 95%confidence intervals (CI) of the geometric mean titer (GMT). Serotype15B data is included to evaluate cross protection.

FIG. 27A-C shows pre (pooled), PD1 (pooled) and PD2 IgG antibodydilution titers as determined by ECL for rabbits immunized with PCV8unadjuvanted, PCV16 unadjuvanted, and PCV31 with APA. Error barsrepresent the 95% confidence intervals (CI) of the geometric mean titer(GMT). Serotype 15B data is included to evaluate cross protection.

FIG. 28 shows the comparison of PD2 ECL antibody responses in NZWR (5per group) following vaccination with PCV21 with or without APA. Symbolsindicate PD2 GMT ratios with error bars representing the 95% CIs.Serotype 15B data is included to evaluate cross protection.

FIG. 29 shows the comparison of PD2 ECL antibody responses in NZWR (5per group) for common serotypes and cross protected serotype 15Bfollowing vaccination with PCV21, PCV8 or PCV16. Symbols indicate PD2ECL GMT ratios with error bars representing the 95% CIs.

FIG. 30 shows the comparison of PD2 ECL antibody responses in NZWR (5per group) for common serotypes and cross protected serotype 15Bfollowing vaccination with PCV21/APA or PCV31/APA. Symbols indicate PD2ECL GMT ratios with error bars representing the 95% CIs.

FIG. 31A-D shows serotype specific Pre (pooled) (A, B) and PD2 (C, D)OPA dilution titers for rabbits immunized with PCVs. Error barsrepresent the 95% CIs of the GMTs. PCV21 was used as the benchmark forstatistical comparisons, *p<0.05. Figures A and C show data for 8 commonserotypes in all PCVs evaluated (6C, 15A, 16F, 23A, 23B, 24F, 31, 35B)and additional 8 common serotypes in PCV16, PCV21 and PCV31 (8, 9N, 10A,11A, 12F, 15C, 17F, 20B). Figures B and D show data for additional 16serotypes not included in Figures A and C. Five of them are also commonserotypes in PCV21 and PCV31 (3, 7F, 19A, 22F, 33F), ten serotypes arecontained exclusively in PCV31 (1, 4, 5, 6A, 6B, 9V, 14, 18C, 19F, 23F).Serotype 15B data is included to evaluate cross protection.

FIG. 32 shows pre, PD1, PD2 and PD3 IgG antibody dilution titers asdetermined by ECL for adult Rhesus macaques (n=8) immunized with PCV21.Error bars represent the 95% CIs of the GMTs. Serotype 15B data isincluded to evaluate cross protection.

FIG. 33 shows pre, PD1 (pooled) and PD3 OPA dilution titers of PCV21immunized adult Rhesus macaques (n=8). Error bars represent the 95% CIsof the GMTs. Serotype 15B data is included to evaluate cross protection.

FIG. 34 shows pre and PD3 (day 70) OPA titers to four serotypes notcontained in the PCV21 vaccine for adult Rhesus macaques immunized withPCV21.

FIG. 35 shows the comparison of PD1 ECL antibody responses in adultRhesus macaques (5 per group) following vaccination with PCV21 with orwithout APA. Symbols indicate PD1 ECL GMT ratios (PCV21 vs. PCV21/APA)with error bars representing the 95% CIs. Serotype 15B data is includedto evaluate cross protection.

FIG. 36 shows PD1 IgG antibody responses to serotype 3, 7F, 19A, 22F,and 33F in adult Rhesus macaques (2-5 per group) following vaccinationwith PCV21 compared to PCV15 or Prevnar13. Symbols indicate PD1 ECL GMTratios with error bars representing the 95% CIs.

FIG. 37 shows ELISA IgG antibody dilution titers to 6A, 6B, and 6C((pre-immune and PD1, pooled) and PD2) for rabbits immunized with6A-CRM197 (A) or 6B-CRM197 (B) monovalent drug products. Bars indicategeometric mean titers (GMTs) and error bars represent the 95% confidenceintervals (CIs) of the GMTs.

FIG. 38 shows serotype specific OPA dilution titers to 6A, 6B, and 6C((pre-immune and PD1 pooled) and PD2) for rabbits immunized with6A-CRM197 (A) or 6B-CRM197 (B) monovalent drug products. Bars indicategeometric mean titers (GMTs) and error bars represent the 95% confidenceintervals (CIs) of the GMTs.

FIG. 39 shows serotype specific OPA dilution titers to 20A and 20B(pre-immune (pooled), PD1 and PD2) for rabbits immunized with20A-CRM197/APA (A) or PCV21 (B). Bars indicate geometric mean titers(GMTs) and error bars represent the 95% confidence intervals (CIs) ofthe GMTs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides multivalent immunogenic compositionscomprising pneumococcal polysaccharide-protein conjugates, wherein eachof the conjugates comprises a polysaccharide from an S. pneumoniaeserotype conjugated to a carrier protein, wherein the serotypes of S.pneumoniae are as defined herein. In some embodiments, the immunogeniccomposition comprises a set of pneumococcal serotypes selected from thegroup consisting of: (a) 15A, 16F, 23A, 23B, 24F, 31 and 35B; (b) 15A,16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20; and(c) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A,11A, 12F, 15C, 17F, and 20. In some embodiments, the immunogeniccomposition comprises a set of pneumococcal serotypes selected from thegroup consisting of: (a) 15A, 16F, 23A, 23B, 24F, 31 and 35B; (b) 15A,16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11 A, 12F, 15C, 17F, and 20B;and (c) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N,10A, 11A, 12F, 15C, 17F, and 20B. In other embodiments, the immunogeniccomposition comprises a set of pneumococcal serotypes selected from thegroup consisting of: (a) 15A, 16F, 23A, 23B, 24F, 31 and 35B; (b) 15A,16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A;and (c) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N,10A, 11 A, 12F, 15C, 17F, and 20A. In further embodiments, theimmunogenic composition comprises (i) serotype 6A; (ii) serotypes 6A and6B; or (iii) serotype 6C. In a particular embodiment, the inventioncomprises multiple pneumococcal S. pneumoniae polysaccharide proteinconjugates wherein each of the conjugates comprises a polysaccharidefrom an S. pneumoniae serotype conjugated to a carrier protein, whereinthe serotypes of S. pneumoniae comprise serotypes 3, 6C, 7F, 8, 9N, 10A,11A, 12F, 15A, 15C, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F and35B. Said composition was found to be immunogenic in rabbits andgenerate functional antibody which killed vaccine-type bacterial strainsat all doses tested (EXAMPLE 43). In another particular embodiment, theinvention comprises multiple pneumococcal S. pneumoniae polysaccharideprotein conjugates wherein each of the conjugates comprises apolysaccharide from an S. pneumoniae serotype conjugated to a carrierprotein, wherein the serotypes of S. pneumoniae comprise serotypes 3,7F, 19A, 22F, 33F, 8, 9N, 10A, 11 A, 12F, 15C, 17F, 20, 6A, 15A, 15C,16F, 23A, 23B, 24F, 31 and 35B. In another particular embodiment, theinvention comprises multiple pneumococcal S. pneumoniae polysaccharideprotein conjugates wherein each of the conjugates comprises apolysaccharide from an S. pneumoniae serotype conjugated to a carrierprotein, wherein the serotypes of S. pneumoniae comprise serotypes 3,7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15C, 17F, 20B, 6A, 15A, 15C,16F, 23A, 23B, 24F, 31 and 35B. In another particular embodiment, theinvention comprises multiple pneumococcal S. pneumoniae polysaccharideprotein conjugates wherein each of the conjugates comprises apolysaccharide from an S. pneumoniae serotype conjugated to a carrierprotein, wherein the serotypes of S. pneumoniae comprise serotypes 3,7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6A, 15A, 15C,16F, 23A, 23B, 24F, 31 and 35B.

The multivalent immunogenic compositions of the invention are useful forimmunizing a patient against vaccine-type S. pneumoniae serotypes and aspart of a treatment regimen with different, complementary pneumococcalvaccine(s). Accordingly, the invention provides a method of inducing aprotective immune response in a human patient comprising administering amultivalent immunogenic composition of the invention to the patient, andfurther comprising administering a multivalent pneumococcal vaccine tothe patient, in any order. In other embodiments, the multivalentimmunogenic compositions of the invention are administered to a patientwho had been previously immunized with a different multivalentpneumococcal vaccine.

In embodiments of the invention, conjugates from at least onepneumococcal serotype are prepared using reductive amination in anaprotic solvent such as DMSO. In further embodiments, the multivalentimmunogenic composition comprises pneumococcal conjugates that were eachprepared using reductive amination in an aprotic solvent. It is shownherein that PCV1, PCV7, PCV14, PCV16 and PCV21 (as defined, infra) drugproducts comprising drug substances that were each prepared usingreductive amination in an aprotic solvent were stable againstdepolymerization or chemical degradation of the carbohydrate and stableagainst aggregation of the protein in the drug product formulation (seeEXAMPLE 40 and EXAMPLE 45). The use of DMSO solvent enhances thecovalent associations of polysaccharide to protein through directconsumption of lysine residues on the surface of the carrier protein.The increased covalent association has a direct benefit to increasingthe stability of the polysaccharide protein conjugate of multivalentimmunogenic compositions comprising polysaccharide antigens conjugatedin DMSO.

I. Definitions and Abbreviations

As used throughout the specification and appended claims, the followingabbreviations apply:

-   -   APA aluminum phosphate adjuvant    -   APC antigen presenting cell    -   CI confidence interval    -   DMSO dimethylsulfoxide    -   DS polysaccharide-protein Drug Substance    -   GMC geometric mean concentration    -   GMT geometric mean titer    -   HPSEC high performance size exclusion chromatography    -   IM intra-muscular or intra-muscularly    -   LOS lipo-oligosaccharide    -   LPS lipopolysaccharide    -   MALS multi-angle light scattering    -   MBC monovalent bulk conjugate    -   MOPA multiplexed opsonophagocytic assays    -   MW molecular weight    -   NMWCO nominal molecular weight cut off    -   NZWR New Zealand White rabbit    -   OPA opsonophagocytosis assay    -   PCV pneumococcal conjugate vaccine    -   PD1 post-dose 1    -   PD2 post-dose 2    -   PnPs Pneumococcal Polysaccharide    -   Ps polysaccharide    -   PS-20 polysorbate-20    -   RI refractive index    -   UV ultraviolet    -   w/v weight per volume

So that the invention may be more readily understood, certain technicaland scientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientificterms used herein have the meaning commonly understood by one ofordinary skill in the art to which this invention belongs.

As used throughout the specification and in the appended claims, thesingular forms “a,” “an,” and “the” include the plural reference unlessthe context clearly dictates otherwise.

Reference to “or” indicates either or both possibilities unless thecontext clearly dictates one of the indicated possibilities. In somecases, “and/or” was employed to highlight either or both possibilities.

The terms “aqueous solvent” or “aqueous conditions” when used withconjugation, such as reductive amination, refers to use of water as thesolvent for the conjugation reaction. The water may contain buffers andother components except that no organic solvent is present.

The terms “aprotic solvent”, “DMSO solvent” or “DMSO conditions” whenused with conjugation, such as reductive amination, refers to use of anaprotic solvent, or a combination of aprotic solvents, (or DMSO, asapplicable) as the solvent for the conjugation reaction. The aproticsolvent may have some water present, for example, up to 1%, 2%, 5%, 10%or 20%.

The term “comprises” when used with the immunogenic composition of theinvention refers to the inclusion of any other components, such asadjuvants and excipients, or the addition of one or morepolysaccharide-protein conjugates that are not specifically enumerated.The term “consisting of” when used with the multivalentpolysaccharide-protein conjugate mixture refers to a mixture havingthose particular S. pneumoniae polysaccharide protein conjugates and noother S. pneumoniae polysaccharide protein conjugates from a differentserotype. “Consists essentially of” and variations such as “consistessentially of” or “consisting essentially of,” indicate the inclusionof any recited elements or group of elements, and the optional inclusionof other elements, of similar or different nature than the recitedelements, which do not materially change the basic or novel propertiesof the specified dosage regimen, method, or composition.

“Effective amount” of a composition of the invention refers to a doserequired to elicit antibodies that significantly reduce the likelihoodor severity of infectivity of a microbe, e.g., S. pneumonia, during asubsequent challenge.

As used herein, the phrase “indicated for the prevention of pneumococcaldisease” means that a vaccine or immunogenic composition is approved byone or more regulatory authorities, such as the US Food and DrugAdministration, for the prophylaxis of one or more diseases caused byany serotype of S. pneumoniae, including, but not limited to:pneumococcal disease generally, pneumococcal pneumonia, pneumococcalmeningitis, pneumococcal bacteremia, invasive disease caused by S.pneumoniae, and otitis media caused by S. pneumoniae.

A “multivalent pneumococcal vaccine” is a pharmaceutical preparationcomprising more than one active agent (e.g., pneumococcal capsularpolysaccharide or pneumococcal polysaccharide protein conjugate) thatprovides active immunity to disease or pathological condition caused bymore than one serotype of S. pneumoniae.

The term “polysaccharide” is meant to include any antigenic saccharideelement (or antigenic unit) commonly used in the immunologic andbacterial vaccine arts, including, but not limited to, a “saccharide”,an “oligosaccharide”, a “polysaccharide”, a “liposaccharide”, a“lipo-oligosaccharide (LOS)”, a “lipopolysaccharide (LPS)”, a“glycosylate”, a “glycoconjugate” and the like.

“PCV1,” as used herein, refers to a 1-valent pneumococcal conjugatevaccine comprising one S. pneumoniae polysaccharide protein conjugate,comprising capsular polysaccharide from a S. pneumoniae serotypeconjugated to a carrier protein, wherein the serotype of S. pneumoniaeis 3 (as exemplified in Example 38 and 45). In specific embodiments, thecarrier protein is CRM197.

“PCV7,” as used herein, refers to a 7-valent pneumococcal conjugatevaccine comprising seven S. pneumoniae polysaccharide proteinconjugates, each comprising capsular polysaccharides from a S.pneumoniae serotype conjugated to a carrier protein, wherein theserotypes of S. pneumoniae are: 3, 8, 9N, 11A, 19A, 15A, and 10A. Inspecific embodiments, the carrier protein of one or more of the S.pneumoniae polysaccharide protein conjugates is CRM197. In furtherembodiments, the carrier protein of each of the S. pneumoniaepolysaccharide protein conjugates is CRM197.

“PCV8,” as used herein, refers to a 8-valent pneumococcal conjugatevaccine comprising eight S. pneumoniae polysaccharide proteinconjugates, each comprising capsular polysaccharide from a S. pneumoniaeserotype conjugated to a carrier protein, wherein the serotypes of S.pneumoniae are: 6C, 15A, 16F, 23A, 23B, 24F, 31, and 35B. In specificembodiments, the carrier protein of one or more of the S. pneumoniaepolysaccharide protein conjugates is CRM197. In further embodiments, thecarrier protein of each of the S. pneumoniae polysaccharide proteinconjugates is CRM197.

“PCV14,” as used herein, refers to a 14-valent pneumococcal conjugatevaccine comprising fourteen S. pneumoniae polysaccharide proteinconjugates, each comprising capsular polysaccharides from a S.pneumoniae serotype conjugated to a carrier protein, wherein theserotypes of S. pneumoniae are: 3, 7F, 8, 9N, 10A, 11A, 12F, 15A, 16F,17F, 19A, 20, 22F and 33F. In specific embodiments, the carrier proteinof one or more of the S. pneumoniae polysaccharide protein conjugates isCRM197. In further embodiments, the carrier protein of each of the S.pneumoniae polysaccharide protein conjugates is CRM197.

“PCV15,” as used herein, refers to a 15-valent pneumococcal conjugatevaccine comprising fifteen S. pneumoniae polysaccharide proteinconjugates, each comprising capsular polysaccharides from a S.pneumoniae serotype conjugated to a carrier protein, wherein theserotypes of S. pneumoniae are: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C,19A, 19F, 22F, 23F and 33F. In specific embodiments, the carrier proteinof one or more of the S. pneumoniae polysaccharide protein conjugates isCRM197. In further embodiments, the carrier protein of each of the S.pneumoniae polysaccharide protein conjugates is CRM197.

“PCV16,” as used herein, refers to a 16-valent pneumococcal conjugatevaccine comprising sixteen S. pneumoniae polysaccharide proteinconjugates, each comprising capsular polysaccharide from a S. pneumoniaeserotype conjugated to a carrier protein, wherein the serotypes of S.pneumoniae are: 6C, 8, 9N, 10A, 11A, 12F, 15A, 16F, 17F, 20A, 23A, 23B,24F, 31, and 35B, and at least one of the following serogroup 15serotypes: 15B, 15C, or de-O-acetylated-15B. In particular embodiments,the serogroup 15 serotype is serotype 15C or de-O-acetylated 15B. Asshown herein (see EXAMPLE 3, infra), de-O-Acetylated serotype 15Bpneumococcal polysaccharide is equivalent to serotype 15C pneumococcalpolysaccharide and has an identical NMR spectra. In specificembodiments, the carrier protein of one or more of the S. pneumoniaepolysaccharide protein conjugates is CRM197. In further embodiments, thecarrier protein of each of the S. pneumoniae polysaccharide proteinconjugates is CRM197.

“PCV21,” as used herein, refers to a 21-valent pneumococcal conjugatevaccine comprising twenty-one S. pneumoniae polysaccharide proteinconjugates, each comprising capsular polysaccharide from a S. pneumoniaeserotype conjugated to a carrier protein, wherein the serotypes of S.pneumoniae are: 3, 6C, 7F, 8, 9N, 10A, 11 A, 12F, 15A, 16F, 17F, 19A,20A, 22F, 23A, 23B, 24F, 31, 33F and 35B, and at least one of thefollowing serogroup 15 serotypes: 15B, 15C or de-O-acetylated-15B. Inparticular embodiments, the serogroup 15 serotype is serotype 15C orde-O-acetylated 15B. In specific embodiments, the carrier protein of oneor more of the S. pneumoniae polysaccharide protein conjugates isCRM197. In further embodiments, the carrier protein of each of the S.pneumoniae polysaccharide protein conjugates is CRM197.

S. pneumoniae serotypes 20A and 20B were identified (Calix, J. J. etal., J. Biol. Chem. (2012) 287(33): 27885-27894) within serogroup 20 in2012 through genetic analysis. As discussed by Calix J. J., et al.,conventional sero-typing methods and commercially available antibodieswere not able to distinguish these two serotypes previously and there islimited information today regarding serotyping efforts for serotypes 20Aand 20B. Further, the disease prevalence of 20A or 20B within anidentified disease caused by serogroup 20 is not well understood. Assuch, one of ordinary skill in the art might elect to include either orboth of these two serotypes (20A and/or 20B) in a S. pneumoniae vaccinecomposition. It is also possible that a S. pneumoniae vaccine containingone serotype 20 polysaccharide antigen might cross protect againstanother (e.g., a vaccine including polysaccharide protein conjugates ofS. pneumoniae serotype 20A, and not serotype 20B, may cross protectagainst infection with S. pneumoniae serotype 20B). Therefore, “serotype20” as used herein may refer to a composition containing polysaccharideprotein conjugates of serotype 20A and/or serotype 20B.

“CpG-containing nucleotide,” “CpG-containing oligonucleotide,” “CpGoligonucleotide,” and similar terms refer to a nucleotide molecule of6-50 nucleotides in length that contains an unmethylated CpG moiety.See, e.g., Wang et al., 2003, Vaccine 21:4297. CpG-containingoligonucleotides include modified oligonucleotides using any syntheticinternucleoside linkages, modified base and/or modified sugar.

An “adjuvant,” as defined herein, is a substance that serves to enhancethe immunogenicity of an immunogenic composition of the invention. Animmune adjuvant may enhance an immune response to an antigen that isweakly immunogenic when administered alone, e.g., inducing no or weakantibody titers or cell-mediated immune response, increase antibodytiters to the antigen, and/or lowers the dose of the antigen effectiveto achieve an immune response in the individual. Thus, adjuvants areoften given to boost the immune response and are well known to theskilled artisan.

A “patient” (alternatively referred to herein as a “subject”) refers toa mammal capable of being infected with a S. pneumoniae. In preferredembodiments, the patient is a human. A patient can be treatedprophylactically or therapeutically. Prophylactic treatment providessufficient protective immunity to reduce the likelihood or severity of apneumococcal infection or the effects thereof, e.g., pneumococcalpneumonia. Therapeutic treatment can be performed to reduce the severityor prevent recurrence of a S. pneumoniae infection or the clinicaleffects thereof. Prophylactic treatment can be performed using amultivalent immunogenic composition of the invention, as describedherein. The composition of the invention can be administered to thegeneral population or to those persons at an increased risk ofpneumococcal infection, e.g. the elderly, or those who live with or carefor the elderly.

Those “in need of treatment” include those previously exposed to orinfected with S. pneumoniae, those who were previously vaccinatedagainst S. pneumoniae, as well as those prone to have an infection orany person in which a reduction in the likelihood of infection isdesired, e.g., the immunocompromised, the elderly, children, adults, orhealthy individuals.

A “stable” multivalent immunogenic composition is a composition whichhas no significant changes observed at a refrigerated temperature (e.g.,2-8° C. or 4° C.) for at least 1 month, 2 months, 3 months, 6 months, 12months and/or 24 months. Additionally, a “stable” composition includesone that exhibits desired features at temperatures including at 25° C.and 37° C. for periods including 1 month, 3 months, 6 months, 12 months,and/or 24 months. Typical acceptable criteria for stability are asfollows: no more than about 5%, about 10%, about 15%, or about 20%variability in one or more of the following: (a) the number averagemolecular weight (Mn) of the S. pneumoniae polysaccharide proteinconjugates in the composition, (b) weight average molecular weight (Mw)of the S. pneumoniae polysaccharide protein conjugates in thecomposition, (c) total polysaccharide concentration in the composition,(d) emission maximum of the composition measured using intrinsic proteinfluorescence spectroscopy at a particular excitation wavelength, e.g.280 nanometers, and (e) the fluorescence intensity of the compositionmeasured using intrinsic protein fluorescence spectroscopy at aparticular excitation wavelength. The term “stable” may also be used torefer to a particular pneumococcal conjugate within a multivalentimmunogenic composition. In such use, the term refers to a conjugatethat exhibits the desired properties over time, at a particulartemperature, and such properties vary no more that about 5%, about 10%,about 15%, or about 20% over the time and temperature noted.

II. Multivalent Immunogenic Compositions

The invention provides multivalent immunogenic compositions comprisingmultiple S. pneumoniae polysaccharide protein conjugates wherein each ofthe conjugates comprises a polysaccharide from an S. pneumoniae serotypeconjugated to a carrier protein. Different aspects and embodiments ofthe multivalent immunogenic compositions of the invention are described,infra.

In one embodiment (Embodiment E1), the invention provides a multivalentimmunogenic composition comprising multiple S. pneumoniae polysaccharideprotein conjugates, each comprising capsular polysaccharide from an S.pneumoniae serotype conjugated to a carrier protein, wherein theserotypes of S. pneumoniae comprise, consist, or consist essentially of:

-   -   (1) 15A,    -   (2) 16F,    -   (3) one or more serogroup 23 serotypes selected from: (a) 23A        and 23B, (b) 23A and 23F, (c) 23B and 23F, (d) 23A, (e) 23B,        and (f) 23F,    -   (4) 24F,    -   (5) 31, and    -   (6) 35B.

In a sub-embodiment of Embodiment E1, the one or more serogroup 23serotypes are 23A and 23B (i.e. and no other serogroup 23 serotypes arepresent). In a further sub-embodiment of Embodiment E1, the one or moreserogroup 23 serotypes are 23A and 23F. In another sub-embodiment ofEmbodiment E1, the one or more serogroup 23 serotypes are 23B and 23F.In yet another sub-embodiment of Embodiment E1, 23A is the onlyserogroup 23 serotype. In a further sub-embodiment of Embodiment E1, 23Bis the only serogroup 23 serotype. In a still further sub-embodiment ofEmbodiment E1, 23F is the only serogroup 23 serotype.

In a second embodiment (Embodiment E2), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates comprising capsular polysaccharidefrom a S. pneumoniae serotype conjugated to a carrier protein, whereinthe serotypes of S. pneumoniae comprise the serotypes set forth inEmbodiment E1, or any sub-embodiment of Embodiment E1, and furthercomprise serotype: (1) 6C, (2) 6A, or (3) 6A and 6B.

In a sub-embodiment of Embodiment E2, the composition comprises serotype6C. In a further sub-embodiment of Embodiment E2, the compositioncomprises serotypes 6A and 6B and does not comprise serotype 6C. In aanother sub-embodiment of Embodiment E2, the composition comprisesserotype 6A.

In a third embodiment (Embodiment E3), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates comprising capsular polysaccharidefrom a S. pneumoniae serotype conjugated to a carrier protein, whereinthe serotypes of S. pneumoniae comprise the serotypes set forth inEmbodiments E1, or any sub-embodiments of Embodiment E1, or EmbodimentE2, or any sub-embodiment of Embodiment E2, and further compriseserotypes:

-   -   (1) 8,    -   (2) 9N,    -   (3) 11A    -   (4) 12F,    -   (5) 15B or 15C,    -   (6) 17F, and    -   (7) 20A and/or 20B.

In a fourth embodiment (Embodiment E4), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates comprising capsular polysaccharidefrom a S. pneumoniae serotype conjugated to a carrier protein, whereinthe serotypes of S. pneumoniae comprise the serotypes set forth in anyof Embodiments E1-E3, or any sub-embodiments thereof, and furthercomprise serotypes: 10A or 39.

In a fifth embodiment (Embodiment E5), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates as described above, wherein the S.pneumoniae serotypes comprise, consist, or consist essentially of: 8,9N, 10A, 11A, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31, and35B.

In a sixth embodiment (Embodiment E6), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates as described above, wherein the S.pneumoniae serotypes comprise, consist, or consist essentially of: 8,9N, 39, 11A, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31, and35B.

In a seventh embodiment (Embodiment E7), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates as described above, wherein the S.pneumoniae serotypes comprise, consist, or consist essentially of: 8,9N, 10A, 11A, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31, and35B.

In an eighth embodiment (Embodiment E8), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates as described above, wherein the S.pneumoniae serotypes comprise, consist, or consist essentially of: 8,9N, 39, 11A, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31, and35B.

In a ninth embodiment (Embodiment E9), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates as described above, wherein the S.pneumoniae serotypes comprise the serotypes set forth in any ofEmbodiments E1-E8 (or any sub-embodiment thereof), and further compriseserotypes: 3, 7F, and 19A.

In a tenth embodiment (Embodiment E10), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates as described above, wherein the S.pneumoniae serotypes comprise the serotypes set forth in any ofEmbodiments E1-E9 (or any sub-embodiment thereof), and further compriseserotype 22F.

In an eleventh embodiment (Embodiment E11), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates as described above, wherein the S.pneumoniae serotypes comprise the serotypes set forth in any ofEmbodiments E1-E10 (or any sub-embodiment thereof), and further compriseserotype 33F.

In an twelfth embodiment (Embodiment E12), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates as described above, wherein the S.pneumoniae serotypes comprise, consist, or consist essentially of a setof S. pneumoniae serotypes selected from the group consisting of:

-   -   (1) 15A, 16F, 23A, 23B, 24F, 31, and 35B,    -   (2) 15A, 16F, 23F, 23B, 24F, 31, and 35B,    -   (3) 15A, 16F, 23A, 23F, 24F, 31, and 35B,    -   (4) 15A, 16F, 23A, 24F, 31, and 35B,    -   (5) 15A, 16F, 23B, 24F, 31, and 35B,    -   (6) 15A, 16F, 23F, 24F, 31, and 35B,    -   (7) 6C, 15A, 16F, 23A, 23B, 24F, 31, and 35B,    -   (8) 6C, 15A, 16F, 23F, 23B, 24F, 31, and 35B,    -   (9) 6C, 15A, 16F, 23A, 23F, 24F, 31, and 35B,    -   (10) 6C, 15A, 16F, 23A, 24F, 31, and 35B,    -   (11) 6C, 15A, 16F, 23B, 24F, 31, and 35B,    -   (12) 6C, 15A, 16F, 23F, 24F, 31, and 35B,    -   (13) 6A, 15A, 16F, 23A, 23B, 24F, 31, and 35B,    -   (14) 6A, 15A, 16F, 23F, 23B, 24F, 31, and 35B,    -   (15) 6A, 15A, 16F, 23A, 23F, 24F, 31, and 35B,    -   (16) 6A, 15A, 16F, 23A, 24F, 31, and 35B,    -   (17) 6A, 15A, 16F, 23B, 24F, 31, and 35B,    -   (18) 6A, 15A, 16F, 23F, 24F, 31, and 35B,    -   (19) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, and 35B,    -   (20) 6A, 6B, 15A, 16F, 23F, 23B, 24F, 31, and 35B,    -   (21) 6A, 6B, 15A, 16F, 23A, 23F, 24F, 31, and 35B,    -   (22) 6A, 6B, 15A, 16F, 23A, 24F, 31, and 35B,    -   (23) 6A, 6B, 15A, 16F, 23B, 24F, 31, and 35B, and    -   (24) 6A, 6B, 15A, 16F, 23F, 24F, 31, and 35B.

In an thirteenth embodiment (Embodiment E13), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates as described above, wherein the S.pneumoniae serotypes comprise, consist, or consist essentially of a setof S. pneumoniae serotypes selected from the group consisting of:

-   -   (1) 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23A, 23B,        24F, 31 and 35B,    -   (2) 8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23A, 23B,        24F, 31 and 35B,    -   (3) 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23A, 23B,        24F, 31 and 35B,    -   (4) 8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23A, 23B,        24F, 31 and 35B,    -   (5) 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 15A, 16F, 23A, 23B,        24F, 31 and 35B,    -   (6) 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6A, 6B, 15A, 16F, 23A,        23B, 24F, 31 and 35B,    -   (7) 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23A, 23B,        24F, 31 and 35B,    -   (8) 8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 15A, 16F, 23A, 23B, 24F,        31 and 35B,    -   (9) 8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 6A, 6B, 15A, 16F, 23A,        23B, 24F, 31 and 35B,    -   (10) 8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23A, 23B,        24F, 31 and 35B,    -   (11) 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 15A, 16F, 23A, 23B,        24F, 31 and 35B,    -   (12) 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6A, 6B, 15A, 16F, 23A,        23B, 24F, 31 and 35B,    -   (13) 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6A, 15A, 16F, 23A,        23B, 24F, 31 and 35B,    -   (14) 8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 15A, 16F, 23A, 23B,        24F, 31 and 35B,    -   (15) 8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 6A, 6B, 15A, 16F, 23A,        23B, 24F, 31 and 35B,    -   (16) 8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 6A, 15A, 16F, 23A, 23B,        24F, 31 and 35B,    -   (17) 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23F,        24F, 31 and 35B,    -   (18) 8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23F, 24F,        31 and 35B,    -   (19) 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23F,        24F, 31 and 35B,    -   (20) 8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23F, 24F,        31 and 35B,    -   (21) 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 15A, 16F, 23F, 24F, 31        and 35B,    -   (22) 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6A, 6B, 15A, 16F, 23F,        24F, 31 and 35B,    -   (23) 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23F,        24F, 31 and 35B,    -   (24) 8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 15A, 16F, 23F, 24F, 31        and 35B,    -   (25) 8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 6A, 6B, 15A, 16F, 23F,        24F, 31 and 35B,    -   (26) 8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23F, 24F,        31 and 35B,    -   (27) 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 15A, 16F, 23F, 24F, 31        and 35B,    -   (28) 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6A, 6B, 15A, 16F, 23F,        24F, 31 and 35B,    -   (29) 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6A, 15A, 16F, 23F,        24F, 31 and 35B,    -   (30) 8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 15A, 16F, 23F, 24F, 31        and 35B,    -   (31) 8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 6A, 6B, 15A, 16F, 23F,        24F, 31 and 35B, and    -   (32) 8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 6A, 15A, 16F, 23F, 24F,        31 and 35B.        The thirteenth embodiment (Embodiment E13), further comprises        the set of S. pneumoniae serotypes, rows (1) through (32)        wherein serotype 20A in each set is substituted with either        serotype 20 or serotype 20B.

In a fourteenth embodiment of the invention, (Embodiment E14), theinvention provides a multivalent immunogenic composition comprisingmultiple S. pneumoniae polysaccharide protein conjugates as describedabove, wherein the S. pneumoniae serotypes comprise, consist, or consistessentially of a set of S. pneumoniae serotypes selected from any of thesets of serotypes set forth in Embodiment E13, rows (17) through (32),and further comprise serotypes 23A and/or 23B.

In an fifteenth embodiment (Embodiment E15), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates as described above, wherein the S.pneumoniae serotypes comprise, consist, or consist essentially of a setof S. pneumoniae serotypes selected from the group consisting of.

-   -   (1) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A,        6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (2) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15C, 17F, 20A,        6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (3) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A,        6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (4) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15B, 17F, 20A,        6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (5) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A        15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (6) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A,        6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (7) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A,        6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (8) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15C, 17F, 20A        15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (9) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15C, 17F, 20A,        6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (10) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15C, 17F, 20A,        6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (11) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A        15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (12) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A,        6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (13) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A,        6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (14) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15B, 17F, 20A        15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (15) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15B, 17F, 20A,        6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (16) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15B, 17F, 20A,        6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   (17) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A,        6C, 15A, 16F, 23F, 24F, 31 and 35B,    -   (18) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15C, 17F, 20A,        6C, 15A, 16F, 23F, 24F, 31 and 35B,    -   (19) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A,        6C, 15A, 16F, 23F, 24F, 31 and 35B,    -   (20) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15B, 17F, 20A,        6C, 15A, 16F, 23F, 24F, 31 and 35B,    -   (21) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A,        15A, 16F, 23F, 24F, 31 and 35B,    -   (22) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A,        6A, 6B, 15A, 16F, 23F, 24F, 31 and 35B,    -   (23) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A,        6A, 15A, 16F, 23F, 24F, 31 and 35B,    -   (24) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15C, 17F, 20A,        15A, 16F, 23F, 24F, 31 and 35B,    -   (25) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15C, 17F, 20A,        6A, 6B, 15A, 16F, 23F, 24F, 31 and 35B,    -   (26) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15C, 17F, 20A,        6A, 15A, 16F, 23F, 24F, 31 and 35B,    -   (27) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A,        15A, 16F, 23F, 24F, 31 and 35B,    -   (28) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A,        6A, 6B, 15A, 16F, 23F, 24F, 31 and 35B,    -   (29) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A,        6A, 15A, 16F, 23F, 24F, 31 and 35B,    -   (30) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15B, 17F, 20A,        15A, 16F, 23F, 24F, 31 and 35B,    -   (31) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15B, 17F, 20A,        6A, 6B, 15A, 16F, 23F, 24F, 31 and 35B,    -   (32) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15B, 17F, 20A,        6A, 15A, 16F, 23F, 24F, 31 and 35B,    -   (33) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20B,    -   (34) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24B, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A,    -   (35) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24B, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20B,    -   (36) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20B,    -   (37) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24B, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A,    -   (38) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24B, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20B,    -   (39) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20,    -   (40) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24B, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20,    -   (41) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24B, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20,    -   (42) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20,    -   (43) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24B, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20, and    -   (44) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24B, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20.

In a sixteenth embodiment of the invention, (Embodiment E16), theinvention provides a multivalent immunogenic composition comprisingmultiple S. pneumoniae polysaccharide protein conjugates as describedabove, wherein the S. pneumoniae serotypes comprise, consist, or consistessentially of a set of S. pneumoniae serotypes selected from any of thesets of serotypes set forth in Embodiment E15, rows (17) through (32),and further comprise serotypes 23A and/or 23B.

In an seventeenth embodiment (Embodiment E17), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates comprising capsular polysaccharidefrom a S. pneumoniae serotype conjugated to a carrier protein, whereinthe serotypes of S. pneumoniae comprise a set of serotypes selected fromthe group consisting of.

-   -   i. 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B,    -   ii. 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F,        15C, 17F, and 20A, and    -   iii. 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A;        In sub-embodiments of Embodiment E17, the immunogenic        composition does not comprise any further S. pneumoniae        polysaccharide protein conjugates.

In an eighteenth embodiment (Embodiment E18), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates comprising capsular polysaccharidefrom a S. pneumoniae serotypes 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B.In sub-embodiments of Embodiment E18, the immunogenic composition doesnot comprise any further S. pneumoniae polysaccharide proteinconjugates.

In a nineteenth embodiment (Embodiment E19), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates comprising capsular polysaccharidefrom a S. pneumoniae serotypes 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8,9N, 10A, 11A, 12F, 15C, 17F, and 20A. In sub-embodiments of EmbodimentE19, the immunogenic composition does not comprise any further S.pneumoniae polysaccharide protein conjugates.

In a twentieth embodiment (Embodiment E20), the invention provides amultivalent immunogenic composition comprising multiple S. pneumoniaepolysaccharide protein conjugates comprising capsular polysaccharidefrom a S. pneumoniae serotypes 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A,23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A. Insub-embodiments of Embodiment E20, the immunogenic composition does notcomprise any further S. pneumoniae polysaccharide protein conjugates.

In another embodiment, the invention provides a multivalent immunogeniccomposition comprising multiple S. pneumoniae polysaccharide proteinconjugates comprising capsular polysaccharide from a S. pneumoniaeserotypes 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8,9N, 10A, 11A, 12F, 15C, 17F, and 20A. In sub-embodiments of thisembodiment (i.e., 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31,35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A), the immunogeniccomposition does not comprise any further S. pneumoniae polysaccharideprotein conjugates.

In another embodiment, the invention provides a multivalent immunogeniccomposition comprising multiple S. pneumoniae polysaccharide proteinconjugates comprising capsular polysaccharide from a S. pneumoniaeserotypes 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8,9N, 10A, 11A, 12F, 15C, 17F, and 20. In sub-embodiments of thisembodiment (i.e., 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31,35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20), the immunogeniccomposition does not comprise any further S. pneumoniae polysaccharideprotein conjugates.

In another embodiment, the invention provides a multivalent immunogeniccomposition comprising multiple S. pneumoniae polysaccharide proteinconjugates comprising capsular polysaccharide from a S. pneumoniaeserotypes 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8,9N, 10A, 11A, 12F, 15C, 17F, and 20B. In sub-embodiments of thisembodiment (i.e., 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31,35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20B), the immunogeniccomposition does not comprise any further S. pneumoniae polysaccharideprotein conjugates.

As shown herein (see EXAMPLE 3, infra), de-O-Acetylated serotype 15Bpneumococcal polysaccharide is equivalent to serotype 15C pneumococcalpolysaccharide and has an identical NMR spectra. De-O-Acetylatedserotype 15B pneumococcal polysaccharide, as described and disclosedherein, is equivalent to serotype 15C pneumococcal polysaccharide andboth may have an O-Acetyl content per repeating unit in the range of0-5%, or in the range of 0-4%, or in the range of 0-3%, or in the rangeof 0-2%, or in the range of 0-1%, or in the range of 0-0.5%, or in therange of 0-0.1%, or no O-Acetyl content. In a report by Spencer B. L.,et al., 15C may be slightly 0-Acetylated (Spencer, B. L. et al., Clin.Vac. Immuno. (2017) 24(8): 1-13). Thus, in any of the embodiments of themultivalent immunogenic compositions herein, de-O-acetylated serotype15B can be used in place of serotype 15C. Processes for de-O-acetylationare known in the art, for example as described in Rajam et al., Clinicaland Vaccine Immunology, 2007, 14(9):1223-1227.

In certain embodiments of any of the multivalent immunogeniccompositions of the invention, including Embodiments E1 to E20 and anysub-embodiment thereof, the composition further comprises apharmaceutically acceptable carrier.

Cross-Reactivity

In an embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugateswherein each of the conjugates comprises a polysaccharide from an S.pneumoniae serotype, including serotype 6C, conjugated to a carrierprotein, wherein serotype 6C of S. pneumoniae provides cross-protectionagainst serotypes 6A and 6B of S. pneumoniae.

In another embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugatesas selected from Embodiments E1 to E20 wherein each of the conjugatescomprises a polysaccharide from an S. pneumoniae serotype, includingserotype 6C, conjugated to a carrier protein, wherein serotype 6C of S.pneumoniae provides cross-protection against serotypes 6A and 6B of S.pneumoniae.

In an embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugateswherein each of the conjugates comprises a polysaccharide from an S.pneumoniae serotype, including serotype 19A, conjugated to a carrierprotein, wherein serotype 19A of S. pneumoniae provides cross-protectionagainst serotype 19F of S. pneumoniae.

In another embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugatesas selected from Embodiments E1 to E20 wherein each of the conjugatescomprises a polysaccharide from an S. pneumoniae serotype, includingserotype 19A, conjugated to a carrier protein, wherein serotype 19A ofS. pneumoniae provides cross-protection against serotype 19F of S.pneumoniae.

In an embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugateswherein each of the conjugates comprises a polysaccharide from an S.pneumoniae serotype, including serotypes 23A and/or 23B, conjugated to acarrier protein, wherein serotypes 23A and/or 23B of S. pneumoniaeprovides cross-protection against serotype 23F of S. pneumoniae.

In another embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugatesas selected from Embodiments E1 to E20 wherein each of the conjugatescomprises a polysaccharide from an S. pneumoniae serotype, includingserotypes 23A and/or 23B, conjugated to a carrier protein, whereinserotypes 23A and/or 23B of S. pneumoniae provides cross-protectionagainst serotype 23F of S. pneumoniae.

In an embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugateswherein each of the conjugates comprises a polysaccharide from an S.pneumoniae serotype, including serotype 6A, conjugated to a carrierprotein, wherein serotype 6A of S. pneumoniae provides cross-protectionagainst serotypes 6B and/or 6C of S. pneumoniae.

In another embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugatesas selected from Embodiments E1 to E20 wherein each of the conjugatescomprises a polysaccharide from an S. pneumoniae serotype, includingserotype 6A, conjugated to a carrier protein, wherein serotype 6A of S.pneumoniae provides cross-protection against serotypes 6B and/or 6C ofS. pneumoniae.

In an embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugateswherein each of the conjugates comprises a polysaccharide from an S.pneumoniae serotype, including serotype 20A, conjugated to a carrierprotein, wherein serotype 20A of S. pneumoniae provides cross-protectionagainst serotype 20B of S. pneumoniae.

In another embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugatesas selected from Embodiments E1 to E20 wherein each of the conjugatescomprises a polysaccharide from an S. pneumoniae serotype, includingserotype 20A, conjugated to a carrier protein, wherein serotype 20A ofS. pneumoniae provides cross-protection against serotype 20B of S.pneumoniae.

In an embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugateswherein each of the conjugates comprises a polysaccharide from an S.pneumoniae serotype, including serotype 20B, conjugated to a carrierprotein, wherein serotype 20B of S. pneumoniae provides cross-protectionagainst serotype 20A of S. pneumoniae.

In another embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugatesas selected from Embodiments E1 to E20 wherein each of the conjugatescomprises a polysaccharide from an S. pneumoniae serotype, includingserotype 20B, conjugated to a carrier protein, wherein serotype 20B ofS. pneumoniae provides cross-protection against serotype 20A of S.pneumoniae.

In an embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugateswherein each of the conjugates comprises a polysaccharide from an S.pneumoniae serotype, including serotype 15C, conjugated to a carrierprotein, wherein serotype 15C of S. pneumoniae provides cross-protectionagainst serotype 15B of S. pneumoniae.

In another embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugatesas selected from Embodiments E1 to E20 wherein each of the conjugatescomprises a polysaccharide from an S. pneumoniae serotype, includingserotype 15C, conjugated to a carrier protein, wherein serotype 15C ofS. pneumoniae provides cross-protection against serotype 15B of S.pneumoniae.

Carrier Protein

In particular embodiments of the present invention, CRM197 is used asthe carrier protein. CRM197 is a non-toxic variant (i.e., toxoid) ofdiphtheria toxin having the following sequence of amino acids.

(SEQ ID NO: 1) JDGGYYGVVN#VIYPHQIVV\#KJWNSJ\YGV#LTNJLTNSNV#JWTJQ\GGGZNHI\VWGQN\#GDDJ\VYGQH#QSOVJNDJJY#YNYW\SJOWN#YODONYGQDHWLNNHOJOVO#WHSOPHTYJW#HHILNUIJGJ#DVUYYOVOSI#DHJVVVYH\LQQZHTDNDOV#YHOHLQIHWU#JNUJTGDP\H#\PDTDFDJQU#YUUVYJVVOVFLQOGZGYLU#GNWNWNLHVO#NHKJSLNQNP#VHVSQNWYVH#HNDNT\OHHIKTWDOHKSHO#VHONWYWJWQ#SYIDJDQ\DD#ZDYQYDTYLG#VHWDGQOHNWWDDOVLOSJL#JVYPJLDGJD#YKKQWHHLYD#TVLDOVVOPY#DTDLSOYJHOYGLJIDD\QI#YHVLLQOITY#YKQV\QUSD\#VSJKNWTSIO#KGJ\DYVZQWYHGVLLUWJI#TJHVJKGLNL#WDHQWSOSLD#JYOOSWLSJN#OGYQNVNWKLVYQJUNLUPU#FUDLGJGYWI#FUSNVSY\YJ#QJYKDQOKYD#IKUVVVHNLKVQHLVVGVLJ#YOJ\TNWYGK#WNYQVNOVOI #IHLNV#

In one embodiment, CRM197 is isolated from cultures of Corynebacteriumdiphtheria strain C7 (β197) grown in casamino acids and yeastextract-based medium. In another embodiment, CRM197 is preparedrecombinantly in accordance with the methods described in U.S. Pat. No.5,614,382. Typically, CRM197 is purified through a combination ofultrafiltration, ammonium sulfate precipitation, and ion-exchangechromatography. In some embodiments, CRM197 is prepared in Pseudomonasfluorescens using Pfenex Expression Technology™ (Pfenex Inc., San Diego,CA).

Other suitable carrier proteins include additional inactivated bacterialtoxins such as DT (Diphtheria toxoid) or fragment B of DT (DTFB), TT(tetanus toxid) or fragment C of TT, pertussis toxoid, cholera toxoid(e.g., as described in WO 2004/083251), E. coli LT, E. coli ST, andexotoxin A from Pseudomonas aeruginosa. Bacterial outer membraneproteins such as outer membrane complex c (OMPC), porins, transferrinbinding proteins, pneumococcal surface protein A (PspA; See WO02/091998), pneumococcal adhesin protein (PsaA), C5a peptidase fromGroup A or Group B streptococcus, or Haemophilus influenzae protein D,pneumococcal pneumolysin (Kuo et al., 1995, Infect Immun 63; 2706-13)including ply detoxified in some fashion for example dPLY-GMBS (See WO04/081515) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE andfusions of Pht proteins for example PhtDE fusions, PhtBE fusions (See WO01/98334 and WO 03/54007), can also be used. Other proteins, such asovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA)or purified protein derivative of tuberculin (PPD), PorB (from N.meningitidis), PD (Haemophilus influenzae protein D; see, e.g., EP 0 594610 B), or immunologically functional equivalents thereof, syntheticpeptides (See EP0378881 and EP0427347), heat shock proteins (See WO93/17712 and WO 94/03208), pertussis proteins (See WO 98/58668 andEP0471177), cytokines, lymphokines, growth factors or hormones (See WO91/01146), artificial proteins comprising multiple human CD4+ T cellepitopes from various pathogen derived antigens (See Falugi et al.,2001, Eur J Immunol 31:3816-3824) such as N19 protein (See Baraldoi etal., 2004, Infect Immun 72:4884-7), iron uptake proteins (See WO01/72337), toxin A or B of C. difficile (See WO 00/61761), and flagellin(See Ben-Yedidia et al., 1998, Immunol Lett 64:9) can also be used ascarrier proteins.

Other DT mutants can be used as the carrier protein, such as CRM₁₇₆,CRM₂₂₈, CRM₄₅ (Uchida et al., 1973, J Biol Chem 218:3838-3844); CRM₉,CRM₄₅, CRM₁₀₂, CRM₁₀₃ and CRM₁₀₇ and other mutations described byNicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, MaecelDekker Inc, 1992; deletion or mutation of Glu-148 to Asp, Gln or Serand/or Ala 158 to Gly and other mutations disclosed in U.S. Pat. No.4,709,017 or 4,950,740; mutation of at least one or more residues Lys516, Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed inU.S. Pat. No. 5,917,017 or 6,455,673; or fragment disclosed in U.S. Pat.No. 5,843,711. Such DT mutants can also be used to make DTFB variantswhere the variants comprise the B fragment contain the epitope regions.

In certain embodiments, the carrier protein is selected from the groupconsisting of: Outer Membrane Protein Complex (OMPC), tetanus toxoid,diphtheria toxoid, protein D and CRM197.

In some embodiments of the invention, a second carrier can be used forone or more of the polysaccharide protein conjugates in the multivalentimmunogenic composition. The second carrier protein is preferably aprotein that is non-toxic and non-reactogenic and obtainable insufficient amount and purity. The second carrier protein is alsoconjugated or joined with the S. pneumoniae polysaccharide to enhanceimmunogenicity of the antigen. Carrier proteins should be amenable tostandard conjugation procedures. In one embodiment, each capsularpolysaccharide not conjugated to the first carrier protein is conjugatedto the same second carrier protein (e.g., each capsular polysaccharidemolecule being conjugated to a single carrier protein). In anotherembodiment, the capsular polysaccharides not conjugated to the firstcarrier protein are conjugated to two or more carrier proteins (eachcapsular polysaccharide molecule being conjugated to a single carrierprotein). In such embodiments, each capsular polysaccharide of the sameserotype is typically conjugated to the same carrier protein.

In embodiments of the invention, including any of Embodiments E1-E20 andany sub-embodiments thereof, one or more (including 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, where applicable) ofthe polysaccharide serotypes is conjugated to CRM197. In furtherembodiments of the invention, including any of Embodiments E1-E20 andany sub-embodiments thereof, each of the polysaccharide serotypes isconjugated to CRM197.

Formulation of the polysaccharide-protein conjugates of the presentinvention can be accomplished using art-recognized methods. Forinstance, individual pneumococcal conjugates can be formulated with aphysiologically acceptable vehicle to prepare the composition. Examplesof such vehicles include, but are not limited to, water, bufferedsaline, polyols (e.g., glycerol, propylene glycol, liquid polyethyleneglycol) and dextrose solutions.

In a preferred embodiment, the vaccine composition is formulated inL-histidine buffer with sodium chloride.

In some embodiments of the invention, the multivalent immunogeniccomposition comprises multiple S. pneumoniae polysaccharide proteinconjugates comprising capsular polysaccharide from an S. pneumoniaeserotype conjugated to a carrier protein and an adjuvant, wherein the S.pneumoniae serotypes are as described herein. Suitable adjuvants toenhance effectiveness of the composition include, but are not limitedto:

(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.;

(2) oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (defined below) orbacterial cell wall components), such as, for example, (a) MF59(International Patent Application Publication No. WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, MA), (b) SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, (c) Ribi™ adjuvant system (RAS), (Corixa,Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components from the group consisting of3-O-deacylated monophosphorylipid A (MPL™) described in U.S. Pat. No.4,912,094, trehalose dimycolate (TDM), and cell wall skeleton (CWS),preferably MPL+CWS (Detox™); and (d) a Montanide ISA;

(3) saponin adjuvants, such as Quil A or STIMULON™ QS-21 (Antigenics,Framingham, MA) (see, e.g., U.S. Pat. No. 5,057,540) may be used orparticles generated therefrom such as ISCOM (immunostimulating complexesformed by the combination of cholesterol, saponin, phospholipid, andamphipathic proteins) and Iscomatrix® (having essentially the samestructure as an ISCOM but without the protein);

(4) bacterial lipopolysaccharides, synthetic lipid A analogs such asaminoalkyl glucosamine phosphate compounds (AGP), or derivatives oranalogs thereof, which are available from Corixa, and which aredescribed in U.S. Pat. No. 6,113,918; one such AGP is2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O—[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside,which is also known as 529 (formerly known as RC529), which isformulated as an aqueous form or as a stable emulsion

(5) synthetic polynucleotides such as oligonucleotides containing CpGmotif(s) (U.S. Pat. No. 6,207,646); and

(6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon),granulocyte macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF),costimulatory molecules B7-1 and B7-2, etc; and

(7) complement, such as a trimer of complement component C3d.

In another embodiment, the adjuvant is a mixture of 2, 3, or more of theabove adjuvants, e.g., SBAS2 (an oil-in-water emulsion also containing3-deacylated monophosphoryl lipid A and QS21).

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

In certain embodiments, the adjuvant is an aluminum salt. The aluminumsalt adjuvant may be an alum-precipitated vaccine or an alum-adsorbedvaccine. Aluminum-salt adjuvants are well known in the art and aredescribed, for example, in Harlow, E. and D. Lane (1988; Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992;Aluminum salts. Research in Immunology 143:489-493). The aluminum saltincludes, but is not limited to, hydrated alumina, alumina hydrate,alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate,alhydrogel, Superfos, Amphogel, aluminum (III) hydroxide, aluminumhydroxyphosphate sulfate, Aluminum Phosphate Adjuvant (APA), amorphousalumina, trihydrated alumina, or trihydroxyaluminum.

APA is an aqueous suspension of aluminum hydroxyphosphate. APA ismanufactured by blending aluminum chloride and sodium phosphate in a 1:1volumetric ratio to precipitate aluminum hydroxyphosphate. After theblending process, the material is size-reduced with a high-shear mixerto achieve a monodisperse particle size distribution. The product isthen diafiltered against physiological saline and steam sterilized.

In certain embodiments, a commercially available Al(OH)₃ (e.g.Alhydrogel or Superfos of Denmark/Accurate Chemical and Scientific Co.,Westbury, NY) is used to adsorb proteins in a ratio of 50-200 μgprotein/mg aluminum hydroxide. Adsorption of protein is dependent, inanother embodiment, on the pI (Isoelectric pH) of the protein and the pHof the medium. A protein with a lower pI adsorbs to the positivelycharged aluminum ion more strongly than a protein with a higher pI.Aluminum salts may establish a depot of antigen that is released slowlyover a period of 2-3 weeks, be involved in nonspecific activation ofmacrophages and complement activation, and/or stimulate innate immunemechanism (possibly through stimulation of uric acid). See, e.g.,Lambrecht et al., 2009, Curr Opin Immunol 21:23.

Monovalent bulk aqueous conjugates are typically blended together anddiluted to target 4 μg/mL for all serotypes except 6B, which will bediluted to target 8 μg/mL. Once diluted, the batch will be filtersterilized, and an equal volume of aluminum phosphate adjuvant addedaseptically to target a final aluminum concentration of 250 μg/mL. Theadjuvanted, formulated batch will be filled into single-use, 0.5 mL/dosevials.

In certain embodiments, the adjuvant is a CpG-containing nucleotidesequence, for example, a CpG-containing oligonucleotide, in particular,a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment,the adjuvant is ODN 1826, which may be acquired from ColeyPharmaceutical Group.

Methods for use of CpG oligonucleotides are well known in the art andare described, for example, in Sur et al., 1999, J Immunol. 162:6284-93;Verthelyi, 2006, Methods Mol Med. 127:139-58; and Yasuda et al., 2006,Crit Rev Ther Drug Carrier Syst. 23:89-110.

In alternative embodiments, the immunogenic composition comprisesmultiple S. pneumoniae polysaccharide protein conjugates as describedherein, for example in any of Embodiments E1-E20 or any sub-embodimentthereof, and does not comprise an adjuvant.

Formulations

The composition of the invention can be formulated as single dose vials,multi-dose vials or as pre-filled glass or plastic syringes.

In another embodiment, compositions of the present invention areadministered orally, and are thus formulated in a form suitable for oraladministration, i.e., as a solid or a liquid preparation. Solid oralformulations include tablets, capsules, pills, granules, pellets and thelike. Liquid oral formulations include solutions, suspensions,dispersions, emulsions, oils and the like.

Pharmaceutically acceptable carriers for liquid formulations are aqueousor nonaqueous solutions, suspensions, emulsions or oils. Examples ofnonaqueous solvents are propylene glycol, polyethylene glycol, andinjectable organic esters such as ethyl oleate. Aqueous carriers includewater, alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Examples of oils are those of animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipidfrom milk or eggs.

The pharmaceutical composition may be isotonic, hypotonic or hypertonic.However it is often preferred that a pharmaceutical composition forinfusion or injection is essentially isotonic, when it is administrated.Hence, for storage the pharmaceutical composition may preferably beisotonic or hypertonic. If the pharmaceutical composition is hypertonicfor storage, it may be diluted to become an isotonic solution prior toadministration.

The isotonic agent may be an ionic isotonic agent such as a salt or anon-ionic isotonic agent such as a carbohydrate. Examples of ionicisotonic agents include but are not limited to NaCl, CaCl₂), KCl andMgCl₂. Examples of non-ionic isotonic agents include but are not limitedto mannitol, sorbitol and glycerol.

It is also preferred that at least one pharmaceutically acceptableadditive is a buffer. For some purposes, for example, when thepharmaceutical composition is meant for infusion or injection, it isoften desirable that the composition comprises a buffer, which iscapable of buffering a solution to a pH in the range of 4 to 10, such as5 to 9, for example 6 to 8.

The buffer may, for example, be selected from the group consisting ofTRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate,citrate, carbonate, glycinate, histidine, glycine, succinate andtriethanolamine buffer.

The buffer may be selected from USP compatible buffers for parenteraluse, in particular, when the pharmaceutical formulation is forparenteral use. For example the buffer may be selected from the groupconsisting of monobasic acids such as acetic, benzoic, gluconic,glyceric and lactic; dibasic acids such as aconitic, adipic, ascorbic,carbonic, glutamic, malic, succinic and tartaric, polybasic acids suchas citric and phosphoric; and bases such as ammonia, diethanolamine,glycine, triethanolamine, and TRIS.

Parenteral vehicles (for subcutaneous, intravenous, intraarterial, orintramuscular injection) include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's and fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Examples are sterile liquids such as water and oils, with orwithout the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. In general, water, saline, aqueous dextrose andrelated sugar solutions, glycols such as propylene glycols orpolyethylene glycol, Polysorbate 80 (PS-80), Polysorbate 20 (PS-20), andPoloxamer 188 (P188) are preferred liquid carriers, particularly forinjectable solutions. Examples of oils are those of animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, olive oil,sunflower oil, fish-liver oil, another marine oil, or a lipid from milkor eggs.

The formulations of the invention may also contain a surfactant.Preferred surfactants include, but are not limited to: thepolyoxyethylene sorbitan esters surfactants (commonly referred to as theTweens), especially PS-20 and PS-80; copolymers of ethylene oxide (EO),propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAX™tradename, such as linear EO/PO block copolymers; octoxynols, which canvary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, withoctoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being ofparticular interest; (octylphenoxy)polyethoxyethanol (IGEPALCA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin);nonylphenol ethoxylates, such as the Tergitol™ NP series;polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl andoleyl alcohols (known as Brij surfactants), such as triethyleneglycolmonolauryl ether (Brij 30); and sorbitan esters (commonly known as theSPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Apreferred surfactant for including in the emulsion is PS-80.

Mixtures of surfactants can be used, e.g. PS-80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (PS-80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as PS-80) 0.01 to 1%, in particular about 0.1%;octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or otherdetergents in the Triton series) 0.001 to 0.1% in particular 0.005 to0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

In certain embodiments, the composition consists essentially ofhistidine (20 mM), saline (150 mM) and 0.2% PS-20 or 0.04% PS-80 at a pHof 5.8 with 250 μg/mL of APA (Aluminum Phosphate Adjuvant). PS-20 canrange from 0.005% to 0.3% (w/v) with the presence of PS-20 or PS-80 informulation controlling aggregation during simulated manufacture and inshipping using primary packaging. In another embodiment, PS-20 can rangefrom 0.025% to 0.8% (w/v). In another embodiment, PS-20 can range from0.05% to 0.8% (w/v). In another embodiment, PS-20 can range from 0.05%to 0.2% (w/v). Process consists of combining a blend of up to 24serotypes in histidine, saline, and PS-20 or PS-80, then combining thisblended material with APA and saline with or without antimicrobialpreservatives.

In particular embodiments, the multivalent immunogenic compositioncomprises S. pneumoniae polysaccharide protein conjugates wherein eachof the conjugates comprises a polysaccharide from an S. pneumoniaeserotype conjugated to a carrier protein, wherein the serotypes of S.pneumoniae in the polysaccharide protein conjugates comprise any of thesets of serotypes set forth herein, and further comprises 20-80 mMhistidine pH 5.8 and 150 mM NaCl. In some embodiments, the multivalentimmunogenic composition further comprises from 0.2% to 0.8% w/vpolysorbate 20.

The choice of surfactant may need to be optimized for different drugproducts and drug substances. For multivalent vaccines having 15 or moreserotypes, PS-20 and P188 are preferred. The choice of chemistry used tomake conjugate can also play an important role in the stabilization ofthe formulation. In particular, when the conjugation reactions used toprepare different polysaccharide protein conjugates in a multivalentcomposition include both aqueous solvent and DMSO solvent, particularsurfactant systems provide significant differences in stability.Improved stability of polysaccharide protein conjugates was seen withpolysorbate 20 alone or with poloxamer 188 in combination with a polyol.

The exact mechanism of how a specific detergent protects abiotherapeutic is poorly understood and cannot be predicted a priori.Possible stabilization mechanisms include preferential hydration,preferential exclusion, air/liquid interface competition betweenbiotherapeutic and surface, surface tension, and/or direct associationof the detergent with the biotherapeutic to mask hydrophobic patcheswhich serve as seeds for aggregation.

Poloxamer may also be used in the compositions of the invention. Apoloxamer is a nonionic triblock copolymer composed of a centralhydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked bytwo hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).Poloxamers are also known by the tradename Pluronic©. Because thelengths of the polymer blocks can be customized, many differentpoloxamers exist that have slightly different properties. For thegeneric term “poloxamer”, these copolymers are commonly named with theletter “P” (for poloxamer) followed by three digits, the first twodigits×100 give the approximate molecular mass of the polyoxypropylenecore, and the last digit×10 gives the percentage polyoxyethylene content(e.g., P407=Poloxamer with a polyoxypropylene molecular mass of 4,000g/mol and a 70% polyoxyethylene content). For the Pluronic© tradename,coding of these copolymers starts with a letter to define its physicalform at room temperature (L=liquid, P=paste, F=flake (solid)) followedby two or three digits. The first digit (two digits in a three-digitnumber) in the numerical designation, multiplied by 300, indicates theapproximate molecular weight of the hydrophobe; and the last digit×10gives the percentage polyoxyethylene content (e.g., L61=Pluronic© with apolyoxypropylene molecular mass of 1,800 g/mol and a 10% polyoxyethylenecontent). See U.S. Pat. No. 3,740,421.

Examples of poloxamers have the general formula:HO(C₂H₄O)_(a)(C₃H₆O)_(b)(C₂H₄O)_(a)H, wherein a and b blocks have thefollowing values:

Pluronic ® Poloxamer A B Molecular Weight L31 2 16 1100 (average) L351900 (average) L44NF 124 12 20 2090 to 2360 L64 2900 (average) L81 2800(average) L121 4400 (average) P123 20 70 5750 (average) F68NF 188 80 277680 to 9510 F87NF 237 64 37 6840 to 8830 F108NF 338 141 44 12700 to17400 F127NF 407 101 56 9840 to 14600 Molecular weight units, as usedherein, are in Dalton (Da) or g/mol.

Preferably, the poloxamer generally has a molecular weight in the rangefrom 1100 to 17,400 Da, from 7,500 to 15,000 Da, or from 7,500 to 10,000Da. The poloxamer can be selected from poloxamer 188 or poloxamer 407.The final concentration of the poloxamer in the formulations is from0.001% to 5% weight/volume, or 0.025% to 1% weight/volume. In certainaspects, the polyol is propylene glycol and is at final concentrationfrom 1% to 20% weight/volume. In certain aspects, the polyol ispolyethylene glycol 400 and is at final concentration from 1% to 20%weight/volume.

Suitable polyols for the formulations of the invention are polymericpolyols, particularly polyether diols including, but are not limited to,propylene glycol and polyethylene glycol, Polyethylene glycol monomethylethers. Propylene glycol is available in a range of molecular weights ofthe monomer from ˜425 to ˜2700. Polyethylene glycol and Polyethyleneglycol monomethyl ether is also available in a range of molecularweights ranging from ˜200 to ˜35000 including but not limited to PEG200,PEG300, PEG400, PEG1000, PEG MME 550, PEG MME 600, PEG MME 2000, PEG MME3350 and PEG MME 4000. A preferred polyethylene glycol is polyethyleneglycol 400. The final concentration of the polyol in the formulations ofthe invention may be 1% to 20% weight/volume or 6% to 20% weight/volume.

The formulation also contains a pH-buffered saline solution. The buffermay, for example, be selected from the group consisting of TRIS,acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate,carbonate, glycinate, histidine, glycine, succinate, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), MES(2-(N-morpholino)ethanesulfonic acid) and triethanolamine buffer. Thebuffer is capable of buffering a solution to a pH in the range of 4 to10, 5.2 to 7.5, or 5.8 to 7.0. In certain aspect of the invention, thebuffer is selected from the group consisting of phosphate, succinate,histidine, MES, MOPS, HEPES, acetate or citrate. The buffer mayfurthermore, for example, be selected from USP compatible buffers forparenteral use, in particular, when the pharmaceutical formulation isfor parenteral use. The concentrations of buffer will range from 1 mM to100 mM. The concentrations of buffer will range from 10 mM to 80 mM. Theconcentrations of buffer will range from 1 mM to 50 mM or 5 mM to 50 mM.In certain aspects, the buffer is histidine at a final concentration of5 mM to 50 mM, or succinate at a final concentration of 1 mM to 10 mM.In certain aspects, the histidine is at a final concentration of 20 mM±2mM.

While the saline solution (i.e., a solution containing NaCl) ispreferred, other salts suitable for formulation include but are notlimited to, CaCl₂), KCl and MgCl₂ and combinations thereof. Non-ionicisotonic agents including but not limited to sucrose, trehalose,mannitol, sorbitol and glycerol may be used in lieu of a salt. Suitablesalt ranges include, but are not limited to 25 mM to 500 mM or 40 mM to170 mM. In one aspect, the saline is NaCl, optionally present at aconcentration from 20 mM to 170 mM.

In a preferred embodiment, the formulations comprise a L-histidinebuffer with sodium chloride.

In another embodiment, the pharmaceutical composition is delivered in acontrolled release system. For example, the agent can be administeredusing intravenous infusion, a transdermal patch, liposomes, or othermodes of administration. In another embodiment, polymeric materials areused; e.g. in microspheres in or an implant.

The amount of conjugate in each vaccine dose is selected as an amountthat induces an immunoprotective response without significant, adverseeffects. Such amount can vary depending upon the pneumococcal serotype.Generally, for polysaccharide-based conjugates, each dose will comprise0.08 to 100 μg of each polysaccharide. In some embodiments of theinvention, the dose of each polysaccharide conjugate is from 0.08 to 10μg. In further embodiments, the dose is from 1 to 5 μg, from 0.4 to 4μg, from 0.4 to 3 μg, from 0.4 to 2 μg, or from 0.4 to 1 μg. In someembodiments, the dose of one or more polysaccharide conjugates is 100,150, 200, 250, 300, 400, 500, or 750 ng or 0.4, 0.5, 0.6, 0.7, 0.75,0.8, 0.9, 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15,16, 18, 20, 22, 25, 30, 40, 50, 60, 70, 80, 90, or 100 ag.

In some embodiments of the compositions of the invention, all of thepolysaccharide conjugates are present in the composition in the sameamount. In further embodiments, the polysaccharide conjugates arepresent in the composition in different amounts (i.e., at least onepolysaccharide conjugate is present in an amount that is different thanone or more of the other polysaccharide conjugates of the composition).

Optimal amounts of components for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. For example, in another embodiment, thedosage for human vaccination is determined by extrapolation from animalstudies to human data. In another embodiment, the dosage is determinedempirically.

In one embodiment, the dose of the aluminum salt is 10, 15, 20, 25, 30,50, 70, 100, 125, 150, 200, 300, 500, or 700 μg, or 1, 1.2, 1.5, 2, 3, 5mg or more. In yet another embodiment, the dose of alum salt describedabove is per μg of recombinant protein.

The compositions of this invention may also include one or more proteinsfrom S. pneumoniae. Examples of S. pneumoniae proteins suitable forinclusion include those identified in International Patent ApplicationPublication Nos. WO 02/083855 and WO 02/053761.

In certain embodiments, the compositions of the invention areadministered to a subject by one or more methods known to a personskilled in the art, such as parenterally, transmucosally, transdermally,intramuscularly, intravenously, intra-dermally, intra-nasally,subcutaneously, intra-peritoneally, and formulated accordingly. In oneembodiment, compositions of the present invention are administered viaepidermal injection, intramuscular injection, intravenous,intra-arterial, subcutaneous injection, or intra-respiratory mucosalinjection of a liquid preparation. Liquid formulations for injectioninclude solutions and the like.

III. Methods of Making

Capsular polysaccharides from Streptococcus pneumoniae can be preparedby standard techniques known to those skilled in the art. For example,polysaccharides can be isolated from bacteria and may be sized to somedegree by known methods (see, e.g., European Patent Nos. EP497524 andEP497525); and preferably by microfluidisation accomplished using ahomogenizer or by chemical hydrolysis. In one embodiment, eachpneumococcal polysaccharide serotype is grown in a soy-based medium. Theindividual polysaccharides are then purified through standard stepsincluding centrifugation, precipitation, and ultra-filtration. See,e.g., U.S. Patent Application Publication No. 2008/0286838 and U.S. Pat.No. 5,847,112. Polysaccharides can be sized in order to reduce viscosityin polysaccharide samples and/or to improve filterability for conjugatedproducts using techniques such as mechanical or chemical sizing.Chemical hydrolysis may be conducted using acetic acid. Mechanicalsizing may be conducted using High Pressure Homogenization Shearing.

The purified polysaccharides can be chemically activated to make thesaccharides capable of reacting with the carrier protein. The purifiedpolysaccharides can be connected to a linker. Once activated orconnected to a linker, each capsular polysaccharide is separatelyconjugated to a carrier protein to form a glycoconjugate. Thepolysaccharide conjugates may be prepared by known coupling techniques.

The polysaccharide can be coupled to a linker to form apolysaccharide-linker intermediate in which the free terminus of thelinker is an ester group. The linker is therefore one in which at leastone terminus is an ester group. The other terminus is selected so thatit can react with the polysaccharide to form the polysaccharide-linkerintermediate.

The polysaccharide can be coupled to a linker using a primary aminegroup in the polysaccharide. In this case, the linker typically has anester group at both termini. This allows the coupling to take place byreacting one of the ester groups with the primary amine group in thepolysaccharide by nucleophilic acyl substitution. The reaction resultsin a polysaccharide-linker intermediate in which the polysaccharide iscoupled to the linker via an amide linkage. The linker is therefore abifunctional linker that provides a first ester group for reacting withthe primary amine group in the polysaccharide and a second ester groupfor reacting with the primary amine group in the carrier molecule. Atypical linker is adipic acid N-hydroxysuccinimide diester (SIDEA).

The coupling can also take place indirectly, i.e. with an additionallinker that is used to derivatise the polysaccharide prior to couplingto the linker.

The polysaccharide is coupled to the additional linker using a carbonylgroup at the reducing terminus of the polysaccharide. This couplingcomprises two steps: (a1) reacting the carbonyl group with theadditional linker; and (a2) reacting the free terminus of the additionallinker with the linker. In these embodiments, the additional linkertypically has a primary amine group at both termini, thereby allowingstep (a1) to take place by reacting one of the primary amine groups withthe carbonyl group in the polysaccharide by reductive amination. Aprimary amine group is used that is reactive with the carbonyl group inthe polysaccharide. Hydrazide or hydroxylamino groups are suitable. Thesame primary amine group is typically present at both termini of theadditional linker. The reaction results in a polysaccharide-additionallinker intermediate in which the polysaccharide is coupled to theadditional linker via a C—N linkage.

The polysaccharide can be coupled to the additional linker using adifferent group in the polysaccharide, particularly a carboxyl group.This coupling comprises two steps: (a1) reacting the group with theadditional linker; and (a2) reacting the free terminus of the additionallinker with the linker. In this case, the additional linker typicallyhas a primary amine group at both termini, thereby allowing step (a1) totake place by reacting one of the primary amine groups with the carboxylgroup in the polysaccharide by EDAC activation. A primary amine group isused that is reactive with the EDAC-activated carboxyl group in thepolysaccharide. A hydrazide group is suitable. The same primary aminegroup is typically present at both termini of the additional linker. Thereaction results in a polysaccharide-additional linker intermediate inwhich the polysaccharide is coupled to the additional linker via anamide linkage.

In one embodiment, the chemical activation of the polysaccharides andsubsequent conjugation to the carrier protein by reductive amination canbe achieved by means described in U.S. Pat. Nos. 4,365,170, 4,673,574and 4,902,506, U.S. Patent Application Publication Nos. 2006/0228380,2007/184072, 2007/0231340 and 2007/0184071, and WO2006/110381,WO2008/079653, and WO2008/143709). The chemistry may include theactivation of pneumococcal polysaccharide by reaction with any oxidizingagent which oxidizes a terminal hydroxyl group to an aldehyde, such asperiodate (including sodium periodate, potassium periodate, or periodicacid). The reaction leads to a random oxidative cleavage of vicinalhydroxyl groups of the carbohydrates with the formation of reactivealdehyde groups.

Coupling to the carrier protein is by reductive amination via directamination to the lysyl groups of the protein. For example, conjugationcan be carried out by reacting a mixture of the activated polysaccharideand carrier protein with a reducing agent such as sodiumcyanoborohydride in the presence of nickel. The conjugation reaction maytake place under aqueous solution or in the presence of DMSO. See, e.g.,US2015/0231270, US2011/0195086 and EP 0471 177 B1. Unreacted aldehydesare then capped with the addition of a strong reducing agent, such assodium borohydride.

Reductive amination involves two steps, (1) oxidation of thepolysaccharide to form reactive aldehydes, (2) reduction of the imine(Schiff base) formed between activated polysaccharide and a carrierprotein to form a stable amine conjugate bond. Before oxidation, thepolysaccharide is optionally size reduced. Mechanical methods (e.g.homogenization) or chemical hydrolysis may be employed. Chemicalhydrolysis may be conducted using acetic acid. The oxidation step mayinvolve reaction with periodate. For the purpose of the presentinvention, the term “periodate” includes both periodate and periodicacid; the term also includes both metaperiodate (IO₄ ⁻) andorthoperiodate (IO₆ ⁻) and includes the various salts of periodate(e.g., sodium periodate and potassium periodate). In an embodiment thecapsular polysaccharide is oxidized in the presence of metaperiodate,preferably in the presence of sodium periodate (NaIO₄). In anotherembodiment the capsular polysaccharide is oxydized in the presence oforthoperiodate, preferably in the presence of periodic acid.

In one embodiment, the oxidizing agent is a stable nitroxyl or nitroxideradical compound, such as piperidine-N-oxy or pyrrolidine-N-oxycompounds, in the presence of an oxidant to selectively oxidize primaryhydroxyls (as described in WO 2014/097099). In said reaction, the actualoxidant is the N-oxoammonium salt, in a catalytic cycle. In an aspect,said stable nitroxyl or nitroxide radical compound are piperidine-N-oxyor pyrrolidine-N-oxy compounds. In an aspect, said stable nitroxyl ornitroxide radical compound bears a TEMPO(2,2,6,6-tetramethyl-1-piperidinyloxy) or a PROXYL(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In an aspect, saidstable nitroxyl radical compound is TEMPO or a derivative thereof. In anaspect, said oxidant is a molecule bearing a N-halo moiety. In anaspect, said oxidant is selected from the group consisting ofN-Chlorosuccinimide, N-Bromosuccinimide, N-Iodosuccinimide,Dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione,Dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione,Diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione.Preferably said oxidant is N-Chlorosuccinimide.

In certain aspects, the oxidizing agent is2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) free radical andN-Chlorosuccinimide (NCS) as the cooxidant (as described in WO2014/097099). Therefore in one aspect, the glycoconjugates from S.pneumoniae are obtainable by a method comprising the steps of a)reacting a saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO)and N-chlorosuccinimide (NCS) in an aqueous solvent to produce anactivated saccharide; and b) reacting the activated saccharide with acarrier protein comprising one or more amine groups (said method isdesignated “TEMPO/NCS-reductive amination” thereafter).

Optionally the oxidation reaction is quenched by addition of a quenchingagent. The quenching agent may be selected from vicinal diols,1-,2-aminoalcohols, amino acids, glutathione, sulfite, bisulfate,dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites orphosphorous acid (such as glycerol, ethylene glycol, propan-1-,2-diol,butan-1,2-diol or butan-2,3-diol, ascorbic acid).

In certain embodiments, the instant invention provides a method forpreparing a serotype 8 Streptococcus pneumoniae polysaccharide-proteinconjugate utilizing a conjugation reaction in an aprotic solvent,wherein the conjugation reaction does not use cyanoborohydride. Infurther embodiments, the conjugation reaction is a Schiff base reductionor reductive amination. In further embodiments, the protein is tetanustoxoid, diphtheria toxoid, or CRM197. In still further embodiments theprotein is CRM197. In further embodiments, the conjugation reaction isreductive amination. In further embodiments, the reductive amination isperformed in dimethylsulfoxide (DMSO).

In some embodiments, the oxidized polysaccharides before conjugationhave a molecular weight of between 30 kDa and 1,000 kDa. Molecularweight can be calculated by size exclusion chromatography (SEC) combinedwith multiangle light scattering detector (MALS) and refractive indexdetector (RI). In some embodiments, the polysaccharide has a molecularweight of between 50 kDa and 300 kDa. In some embodiments, thepolysaccharide has a molecular weight of between 50 kDa and 1,000 kDa.In additional embodiments, the polysaccharide has a molecular weight ofbetween 70 kDa and 900 kDa. In other embodiments, the polysaccharide hasa molecular weight of between 100 kDa and 800 kDa. In other embodiments,the polysaccharide has a molecular weight of between 200 kDa and 600kDa. In further embodiments, the polysaccharide has a molecular weightof 100 kDa to 1,000 kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa; 100 kDato 700 kDa; 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa;100 kDa to 300 kDa; 150 kDa to 1,000 kDa; 150 kDa to 900 kDa; 150 kDa to800 kDa; 150 kDa to 700 kDa; 150 kDa to 600 kDa; 150 kDa to 500 kDa; 150kDa to 400 kDa; 150 kDa to 300 kDa; 200 kDa to 1,000 kDa; 200 kDa to 900kDa; 200 kDa to 800 kDa; 200 kDa to 700 kDa; 200 kDa to 600 kDa; 200 kDato 500 kDa; 200 kDa to 400 kDa; 200 kDa to 300; 250 kDa to 1,000 kDa;250 kDa to 900 kDa; 250 kDa to 800 kDa; 250 kDa to 700 kDa; 250 kDa to600 kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300kDa to 1,000 kDa; 300 kDa to 900 kDa; 300 kDa to 800 kDa; 300 kDa to 700kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDato 1,000 kDa; 400 kDa to 900 kDa; 400 kDa to 800 kDa; 400 kDa to 700kDa; 400 kDa to 600 kDa; 500 kDa to 600 kDa.

The second step of the conjugation process is the reduction of the imine(Schiff base) bond between activated polysaccharide and a carrierprotein to form a stable conjugate bond (so-called reductive amination),using a reducing agent. Reducing agents which are suitable include thecyanoborohydrides (such as sodium cyanoborohydride or sodiumborohydride). In one embodiment the reducing agent is sodiumcyanoborohydride.

In certain embodiments, the reductive amination reaction is carried outin aprotic solvent (or a mixture of aprotic solvents). In oneembodiment, the reduction reaction is carried out in DMSO or in DMF(dimethylformamide) solvent. The DMSO or DMF solvent may be used toreconstitute the activated polysaccharide and carrier protein, iflyophilized. In one embodiment, the aprotic solvent is DMSO.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, which may be capped using a suitablecapping agent. In one embodiment this capping agent is sodiumborohydride (NaBH₄). Suitable alternatives include sodiumtriacetoxyborohydride or sodium or zinc borohydride in the presence ofBronsted or Lewis acids), amine boranes such as pyridine borane,2-Picoline Borane, 2,6-diborane-methanol, dimethylamine-borane,t-BuMe′PrN—BH₃, benzylamine-BH₃ or 5-ethyl-2-methylpyridine borane(PEMB) or borohydride exchange resin. Following the conjugation (thereduction reaction and optionally the capping), the glycoconjugates maybe purified (enriched with respect to the amount ofpolysaccharide-protein conjugate) by a variety of techniques known tothe skilled person. These techniques include dialysis,concentration/diafiltration operations, tangential flow filtration,precipitation/elution, column chromatography (ion exchangechromatography, multimodal ion exchange chromatography, DEAE, orhydrophobic interaction chromatography), and depth filtration. In anembodiment, the glycoconjugates are purified by diafilitration or ionexchange chromatography or size exclusion chromatography.

Glycoconjugates prepared using reductive amination in an aprotic solventare generally used in multivalent pneumococcal conjugate vaccines. Thus,in certain embodiments for multivalent compositions where not all theserotypes are prepared in an aprotic solvent, the reduction reaction forthe remaining seroytpes is carried out in aqueous solvent (e.g.,selected from PBS (phosphate buffered saline), MES(2-(N-morpholino)ethanesulfonic acid), HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Bis-tris, ADA(N-(2-Acetamido)iminodiacetic acid), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)), MOPSO(3-Morpholino-2-hydroxypropanesulfonic acid), BES(N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), DIPSO (3-Bis(2-hydroxyethyl)amino-2-hydroxypropane-1-sulfonic acid), MOBS(4-(N-morpholino)butanesulfonic acid), HEPPSO(N-(2-Hydroxyethyl)piperazine-N-(2-hydroxypropanesulfonic acid)), POPSO(Piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid)), TEA(triethanolamine), EPPS (4-(2-Hydroxyethyl)piperazine-1-propanesulfonicacid), Bicine at a pH between 6.0 and 8.5, 7.0 and 8.0, or 7.0 and 7.5).

S. pneumonia capsular polysaccharide-protein conjugates that can beprepared using reductive amination in an aprotic solvent, include, butare not limited to, serotypes: 3, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F,15A, 15B, 15C, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 23F, 24F, 31, 33F,35B, and 39. S. pneumonia capsular polysaccharide-protein conjugatesthat can be prepared using reductive amination in an aprotic solvent,include, but are not limited to, serotypes: 3, 6A, 6B, 6C, 7F, 8, 9N,10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 19A, 20, 22F, 23A, 23B, 23F,24F, 31, 33F, 35B, and 39. S. pneumonia capsular polysaccharide-proteinconjugates that can be prepared using reductive amination in an aproticsolvent, include, but are not limited to, serotypes: 3, 6A, 6B, 6C, 7F,8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 19A, 20B, 22F, 23A, 23B,23F, 24F, 31, 33F, 35B, and 39. The polysaccharides may be used in theform of oligosaccharides. These are conveniently formed by fragmentationof purified polysaccharide (e.g. by hydrolysis), which will usually befollowed by purification of the fragments of the desired size.

In certain embodiments, pneumococcal polysaccharide-protein conjugatesof one or more of serotypes 3, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F,15A, 15B, 15C, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 23F, 24F, 31, 33F,35B, and 39 are prepared using reductive amination in an aproticsolvent. In certain embodiments, pneumococcal polysaccharide-proteinconjugates of one or more of serotypes 3, 6A, 6B, 6C, 7F, 8, 9N, 10A,11A, 12F, 15A, 15B, 15C, 16F, 17F, 19A, 20, 22F, 23A, 23B, 23F, 24F, 31,33F, 35B, and 39 are prepared using reductive amination in an aproticsolvent. In certain embodiments, pneumococcal polysaccharide-proteinconjugates of one or more of serotypes 3, 6A, 6B, 6C, 7F, 8, 9N, 10A,11A, 12F, 15A, 15B, 15C, 16F, 17F, 19A, 20B, 22F, 23A, 23B, 23F, 24F,31, 33F, 35B, and 39 are prepared using reductive amination in anaprotic solvent. In certain embodiments, each of the serotypes in themultivalent immunogenic composition is prepared using reductiveamination in an aprotic solvent. In certain embodiments, polysaccharidesof one or more serotypes in a multivalent composition are conjugatedusing reductive amination in an aprotic solvent and polysaccharides ofone or more serotypes are conjugated using reductive amination in anaqueous solvent. In certain embodiments, polysaccharides of two or moreserotypes in a multivalent composition are conjugated using reductiveamination in an aprotic solvent. In other embodiments, polysaccharidesof three or more, four or more, five or more, six or more, seven ormore, eight or more, nine or more, ten or more, eleven or more, twelveor more, thirteen or more, fourteen or more, fifteen or more, sixteen ormore, seventeen or more, eighteen or more, nineteen or more, twenty ormore, or twenty-one or more serotypes in a multivalent composition areconjugated using reductive amination in an aprotic solvent. In certainembodiments, polysaccharides from one or more serotypes in a multivalentcomposition are conjugated using other chemistries which may be in anaprotic solvent or in an aqueous solvent.

Thus, the invention relates to a multivalent immunogenic compositioncomprising multiple S. pneumoniae polysaccharide protein conjugates,each comprising capsular polysaccharide from an S. pneumoniae serotypeconjugated to a carrier protein, wherein the serotypes of S. pneumoniaeare as described herein (i.e. in Section II, “Multivalent ImmunogenicCompositions”), wherein the conjugation reaction whereby thepolysaccharide from one or more of the polysaccharide protein conjugatesis conjugated to the carrier protein is in an aprotic solvent. Incertain embodiments, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100% of the serotypes in a multivalent compositionare prepared in an aprotic solvent. The remainder of the serotypes areprepared using an alternative chemistry and/or in an aqueous solvent.

It was determined that the use of DMSO as a solvent during reductiveamination of polysaccharide-protein conjugates results in theunexpectedly superior stability and enhanced immunogenicity for thoseserotypes relative to the same conjugates prepared under aqueousconditions (See U.S. Application Ser. Nos. 62/463,216 and 62/555,444).As shown herein (see EXAMPLE 40), drug product formulations containingthe pneumococcal conjugates of the invention prepared using reductiveamination in an aprotic solvent (e.g. DMSO) resulted in superiorphysical and chemical stability as compared to a vaccine utilizing drugsubstances prepared using a protic (i.e. aqueous) solvent duringreductive amination in the conjugation process. Thus, in someembodiments, all of the pneumococcal polysaccharide conjugates in themultivalent composition are prepared in an aprotic solvent.

In certain embodiments of the invention the total polysaccharideconcentration in the composition is from about 0.02 to about 0.175mg/mL. In certain embodiments of the invention the total polysaccharideconcentration in the composition is from about 0.03 to about 0.175mg/mL. In certain embodiments of the invention the total polysaccharideconcentration in the composition is from about 0.04 to about 0.175mg/mL. In other embodiments, the total polysaccharide concentration inthe composition is from about 0.065 to about 0.085 mg/mL, about 0.070 toabout 0.080 mg/mL, about 0.065 to about 0.080 mg/mL, about 0.070 toabout 0.085 mg/mL, about 0.110 to about 0.128 mg/mL, about 0.110 toabout 0.175 mg/mL, about 0.10 to about 0.175 mg/mL, about 0.110 to about0.170 mg/mL, about 0.115 to about 0.15 mg/mL, about 0.110 to about 0.15mg/mL, about 0.110 to about 0.125 mg/mL, about 0.150 to about 0.170mg/mL, about 0.150 to about 0.165 mg/mL, about 0.140 to about 0.170mg/mL, about 0.130 to about 0.170 mg/mL, about 0.150 to about 0.175mg/mL, about 0.070 to about 0.170 mg/mL, about 0.065 to about 0.175mg/mL, or about 0.065 to about 0.180 mg/mL.

In embodiments of the invention wherein one or more, or all, of thepolysaccharide-protein conjugates in the multivalent immunogeniccompositions are prepared in an aprotic solvent, the totalpolysaccharide concentration in the composition is stable for 4 weeks ormore at 37° C., 4 weeks or more at 25° C., and 12 weeks or more at 4° C.

In certain embodiments of the invention wherein one or more, or all, ofthe polysaccharide-protein conjugates in the multivalent immunogeniccompositions are prepared in an aprotic solvent, the weight averagemolecular weight (Mw) of all of the S. pneumoniae polysaccharide proteinconjugates in the composition (average of all conjugates in thecomposition) is from about 3,500 to about 4,700 kDa, from about 3,500 toabout 4,600 kDa, from about 3,500 to about 4,500 kDa, from about 3,500to about 4,400 kDa, from about 3,500 to about 4,300 kDa, from about3,500 to about 4,200 kDa, from about 3,600 to about 4,700 kDa, fromabout 3,600 to about 4,600 kDa, from about 3,600 to about 4,500 kDa,from about 3,600 to about 4,400 kDa, from about 3,600 to about 4,300kDa, from about 3,600 to about 4,200 kDa, from about 3,700 to about4,700 kDa, from about 3,700 to about 4,600 kDa, from about 3,700 toabout 4,500 kDa, from about 3,700 to about 4,400 kDa, from about 3,700to about 4,300 kDa, from about 3,700 to about 4,200 kDa, from about3,800 to about 4,700 kDa, from about 3,800 to about 4,600 kDa, fromabout 3,800 to about 4,500 kDa, from about 3,800 to about 4,400 kDa,from about 3,800 to about 4,300 kDa, from about 3,800 to about 4,200kDa, from about 3,900 to about 4,700 kDa, from about 3,900 to about4,600 kDa, from about 3,900 to about 4,500 kDa, from about 3,900 toabout 4,400 kDa, from about 3,900 to about 4,300 kDa, or from about3,900 to about 4,200 kDa.

In certain embodiments of the invention wherein thepolysaccharide-protein conjugates in the multivalent immunogeniccompositions are prepared in an aprotic solvent, the Mw of each of theS. pneumoniae polysaccharide protein conjugates in the composition (fora single serotype) is from about 1,000 to about 10,000 kDa, from about1,500 to about 5,500 kDa, from about 1,500 to about 5,600 kDa, fromabout 1,500 to about 5,700 kDa, from about 1,500 to about 5,800 kDa,from about 1,500 to about 5,900 kDa, from about 1,500 to about 6,000kDa, from about 1,000 to about 5,500 kDa, from about 1,000 to about5,000 kDa, from about 1,000 to about 4,000 kDa, from about 1,000 toabout 4,500 kDa, from about 1,000 to about 4,000 kDa, or from about1,000 to about 3,500 kDa. In other embodiments, the Mw of a conjugatefrom a single serotype within the composition is about 1,000 kDa, about1,100 kDa, about 1,200 kDa, about 1,300 kDa, about 1,400 kDa, about1,500 kDa, about 1,600 kDa, about 1,700 kDa, about 1,800 kDa, about1,900 kDa, about 2,000 kDa, about 2,100 kDa, about 2,200 kDa, about2,300 kDa, about 2,400 kDa, about 2,500 kDa, about 2,600 kDa, about2,700 kDa, about 2,800 kDa, about 2,900 kDa, about 3,000 kDa, about3,100 kDa, about 3,200 kDa, about 3,300 kDa, about 3,400 kDa, about3,500 kDa, about 3,600 kDa, about 3,700 kDa, about 3,800 kDa, about3,900 kDa, about 4,000 kDa, about 4,100 kDa, about 4,200 kDa, about4,300 kDa, about 4,400 kDa, about 4,500 kDa, about 4,600 kDa, about4,700 kDa, about 4,800 kDa, about 4,900 kDa, about 5,000 kDa, about5,100 kDa, about 5,200 kDa, about 5,300 kDa, about 5,400 kDa, or about5,500 kDa.

In certain embodiments of the invention the polysaccharide-proteinconjugates in the multivalent immunogenic compositions are prepared inan aprotic solvent. Compositions containing higher percentages of S.pneumoniae polysaccharides conjugated to a carrier protein in an aproticsolvent (as opposed to being prepared in a protic solvent) may bepreferred. In certain embodiments, the percentage (as calculated by thenumber of polysaccharide serotypes prepared in an aprotic solventdivided by the total number of polysaccharide serotypes, where totalnumber includes those prepared in an aprotic solvent or a proticsolvent) of S. pneumoniae serotype specific conjugates prepared in anaprotic solvent may be greater than 50%, or greater than 60%, or greaterthan 70%, or greater than 80%, or greater than 90% or are 100%.

In certain embodiments of the invention, the serotype 3polysaccharide-protein conjugate in the composition is prepared in anaprotic solvent and the Mw of said conjugate is from about 1,000 toabout 5,000 kDa, or from about 1,000 to about 4,000 kDa, or from about1,000 to about 3,000 kDa, or from about 1,000 to about 2,500 kDa, orfrom about 1,000 to about 2,000 kDa.

In certain embodiments of the invention wherein one or more, or all, ofthe polysaccharide-protein conjugates in the multivalent immunogeniccompositions are prepared in an aprotic solvent, the number averagemolecular weight (Mn) of the S. pneumoniae polysaccharide proteinconjugates in the composition (average of all conjugates in thecomposition) is from about 900 to about 3,000 kDa, from about 1,000 toabout 3,000 kDa, from about 1,000 to about 2,500 kDa, from about 1,500to about 2,500 kDa, from about 1,800 to about 2,500 kDa, from about1,900 to about 2,500 kDa, or from about 2,000 to about 2,500 kDa.

In certain embodiments of the invention wherein one or more, or all, ofthe polysaccharide-protein conjugates in the multivalent immunogeniccompositions are prepared in an aprotic solvent, the Mn of each of theS. pneumoniae polysaccharide protein conjugates in the composition (fora single serotype) is from about 700 to about 7,000 kDa, from about1,000 to about 6,000 kDa, from about 1,000 to about 5,000 kDa, fromabout 1,000 to about 4,000 kDa, from about 1,000 to about 3,000 kDa,from about 900 to about 5,500 kDa, from about 900 to about 5,000 kDa,from about 900 to about 4,500 kDa, from about 900 to about 4,000 kDa,from about 900 to about 3,500 kDa, or from about 900 to about 3,000 kDa.

In embodiments of the invention, the Mw and/or Mn of the S. pneumoniaepolysaccharide protein conjugates in the composition is stable for 4weeks or more at 37° C., 4 weeks or more at 25° C., and/or 12 weeks ormore at 4° C.

In embodiments of the invention, the polysaccharide concentration, Mw,and/or Mn are determined using HPSEC UV/MALS/RI.

In some embodiment of the invention, wherein one or more, or all, of thepolysaccharide-protein conjugates in the multivalent immunogeniccompositions are prepared in an aprotic solvent, the emission maximum ofthe composition measured using intrinsic protein fluorescencespectroscopy with an excitation wavelength at 280 nanometers (nm) isfrom about 335 nm to about 342 nm. In some embodiments, the emissionmaximum remains from about 335 nm to about 342 nm and the fluorescenceintensity is stable for at least 1 week at 37° C. In some embodiments,the emission maximum remains from about 335 nm to about 342 nm and thefluorescence intensity is stable for 1 week at 37° C.

In some embodiments, all of the pneumococcal polysaccharide conjugatesin the multivalent composition are prepared using reductive amination inDMSO. In certain sub-embodiments, the multivalent composition comprisingpolysaccharide conjugates which were all prepared using DMSO does notcomprise an adjuvant.

Without being bound by any theory, one possible mechanism for theenhanced immunogenicity observed with glycoconjugates prepared in DMSOinclude an increased number of linkages between the carbohydrate(capsular polysaccharide) and lysine residues on the surface of thecarrier protein which would result in additional attachment pointsbetween the protein and polysaccharide to impart stability and counterchemical depolymerization or breakdown of the peptide carbohydrate bond.See, e.g., Hsieh, Characterization of Saccharide-CRM197 ConjugateVaccines in Brown F, Corbel M, Griffiths E (eds): Physico-ChemicalProcedures for the Characterization of Vaccines. Dev. Biol. Basel,Karger, 2000, vol 103, pp. 93-104. An additional benefit of theincreased polysaccharide-protein linkages that are created duringconjugation in the DMSO solvent could be additional opportunities forsuccessful presentation of peptide-carbohydrate to T-cells. It can beappreciated that due to the genetic variability in the human populationresulting in varying abilities and sensitivity of loading or associatingwith specific peptide sequences conjugated to carbohydrate antigens,that additional points of attachment on the carrier protein would allowfor increased chances for successful antigen presentation at the surfaceof an antigen presenting cell (APC) to allow for a T-cell dependentresponse to an otherwise T-cell independent antigen. Another possiblemechanism of enhanced immunogenicity observed by conjugation in the DMSOsolvent could be due to the denaturation of CRM197 in organic solvent,which exposes additional lysines for polysaccharide linkages givingincreased chances for glycopeptide presentation at the surface of an APCfor T-cell dependent response to different peptide epitopes. See Avci etal., 2011, Nature Medicine 17: 1602-1610.

Yet another benefit of conjugation in an organic solvent generatingdenatured CRM197 in the conjugates could be reduced immunologicalinterference of antibodies against native CRM197 epitopes. A furtherbenefit of the increased polysaccharide-protein linkages that arecreated during conjugation in the DMSO solvent could be the formation oflarger sized polysaccharide protein conjugates resulting in enhancedimmunogenicity. The compositions of the invention are believed toprovide significant advantages in eliciting a human response.

In certain embodiments, the conjugation reaction is performed byreductive amination wherein nickel is used for greater conjugationreaction efficiency and to aid in free cyanide removal. Transitionmetals are known to form stable complexes with cyanide and are known toimprove reductive methylation of protein amino groups and formaldehydewith sodium cyanoborohydride (S Gidley et al., Biochem J 1982, 203:331-334; Jentoft et al. Anal Biochem. 1980, 106: 186-190). By complexingresidual, inhibitory cyanide, the addition of nickel increases theconsumption of protein during the conjugation and leads to formation oflarger, potentially more immunogenic conjugates.

Differences in starting cyanide levels in sodium cyanoborohydridereagent lots also lead to inconsistent conjugation performance,resulting in variable product attributes, such as conjugate size andconjugate Ps-to-CRM197 ratio. The addition of nickel reduced conjugationinconsistency by complexing cyanide, eliminating differences in sodiumcyanoborohydride lots.

Suitable alternative chemistries include the activation of thesaccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP) to form a cyanate ester. The activated saccharide may thus becoupled directly or via a spacer (linker) group to an amino group on thecarrier protein. For example, the spacer could be cystamine or cystamineto give a thiolated polysaccharide which could be coupled to the carriervia a thioether linkage obtained after reaction with amaleimide-activated carrier protein (for example using GMBS) or ahaloacetylated carrier protein (for example using iodoacetimide [e.g.ethyl iodoacetimide HCl] or N-succinimidyl bromoacetate or SIAB, or SIA,or SBAP). Preferably, the cyanate ester (optionally made by CDAPchemistry) is coupled with hexane diamine or adipic acid dihydrazide(ADH) and the amino-derivatised saccharide is conjugated to the carrierprotein using carbodiimide (e.g. EDAC or EDC) chemistry via a carboxylgroup on the protein carrier. Such conjugates are described inInternational Patent Application Publication Nos. WO 93/15760, WO95/08348 and WO 96/29094; and Chu et al., 1983, Infect. Immunity40:245-256.

Other suitable techniques use carbodiimides, hydrazides, active esters,norbornane, p-nitrobenzoic acid, N-hydroxysuccinimide, S—NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(See Bethell et al., 1979, J. Biol. Chem. 254:2572-4; Hearn et al.,1981, J. Chromatogr. 218:509-18) followed by reaction of with a proteinto form a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

After conjugation of the capsular polysaccharide to the carrier protein,the polysaccharide-protein conjugates are purified (enriched withrespect to the amount of polysaccharide-protein conjugate) by one ormore of a variety of techniques. Examples of these techniques are wellknown to the skilled artisan and include concentration/diafiltrationoperations, ultrafiltration, precipitation/elution, columnchromatography, and depth filtration. See, e.g., U.S. Pat. No.6,146,902.

After the individual glycoconjugates are purified, they are compoundedto formulate the immunogenic composition of the present invention. Thesepneumococcal conjugates are prepared by separate processes and bulkformulated into a single dosage formulation.

An alternative method for characterizing the glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM197) that become conjugated to the saccharide which can becharacterized as a range of conjugated lysines (degree of conjugation).The evidence for lysine modification of the carrier protein, due tocovalent linkages to the polysaccharides, can be obtained by amino acidanalysis using routine methods known to those of skill in the art.Conjugation results in a reduction in the number of lysine residuesrecovered, compared to the carrier protein starting material used togenerate the conjugate materials. In a preferred embodiment, the degreeof conjugation of the glycoconjugate of the invention is between 2 and15, between 2 and 13, between 2 and 10, between 2 and 8, between 2 and6, between 2 and 5, between 2 and 4, between 3 and 15, between 3 and 13,between 3 and 10, between 3 and 8, between 3 and 6, between 3 and 5,between 3 and 4, between 5 and 15, between 5 and 10, between 8 and 15,between 8 and 12, between 10 and 15 or between 10 and 12. In anembodiment, the degree of conjugation of the glycoconjugate of theinvention is about 2, about 3, about 4, about 5, about 6, about 7, about8, about 9, about 10, about 11, about 12, about 13, about 14 or about15. In a preferred embodiment, the degree of conjugation of theglycoconjugate of the invention is between 4 and 7. In some suchembodiments, the carrier protein is CRM197.

The glycoconjugates of the compositions of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein (Ps:Pr). In some embodiments, the ratio of polysaccharide tocarrier protein of the glycoconjugates (w/w) in the composition isbetween 0.5 and 3.0 (e.g., about 0.5, about 0.6, about 0.7, about 0.8,about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1,about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about2.8, about 2.9, or about 3.0). In other embodiments, the saccharide tocarrier protein ratio (w/w) is between 0.5 and 2.5, between 0.5 and 1.5,between 0.8 and 2.5, between 0.5 and 1.0, between 1.0 and 1.5, between1.0 and 2.0, between 0.8 and 2.4, between 0.8 and 2.3, between 0.8 and2.2, between 0.8 and 2.1, between 0.8 and 2.0, between 0.8 and 1.9,between 0.8 and 1.8, between 0.8 and 1.7, between 0.8 and 1.6, between0.8 and 1.5, between 0.8 and 1.4, between 0.8 and 1.3, between 0.9 and2.4, between 0.9 and 2.3, between 0.9 and 2.2, between 0.9 and 2.1,between 0.9 and 2.0, between 0.9 and 1.9, between 0.9 and 1.8, between0.9 and 1.7, between 0.9 and 1.6, between 0.9 and 1.5, between 0.9 and1.4, between 0.9 and 1.3, between 0.9 and 1.2, between 1.0 and 2.4,between 1.0 and 2.3, between 1.0 and 2.2, between 1.0 and 2.1, between1.0 and 2.0, between 1.0 and 1.9, between 1.0 and 1.8, between 1.0 and1.7, between 1.0 and 1.6, between 1.0 and 1.5, between 1.0 and 1.4,between 1.0 and 1.3 or between 1.0 and 1.2. In further embodiments, thesaccharide to carrier protein ratio (w/w) is between 0.8 and 1.2. Insome such embodiments, the carrier protein is CRM197. Theglycoconjugates and immunogenic compositions of the invention maycontain free saccharide that is not covalently conjugated to the carrierprotein, but is nevertheless present in the glycoconjugate composition.The free saccharide may be non-covalently associated with (i.e.,non-covalently bound to, adsorbed to, or entrapped in or with) theglycoconjugate.

In specific embodiments, the saccharide to carrier protein ratio (w/w)for the serotype 15A conjugate is from about 1.0 to about 2.0, fromabout 1.25 to about 1.75, or from about 1.3 to about 1.7. In otherembodiments, the saccharide to carrier protein ratio (w/w) for serotype15A is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8.

In specific embodiments, the saccharide to carrier protein ratio (w/w)for the serotype 15C conjugate is from about 1.0 to about 2.0, fromabout 1.25 to about 1.75, or from about 1.3 to about 1.7. In otherembodiments, the saccharide to carrier protein ratio (w/w) for serotype15C is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8.

In specific embodiments, the saccharide to carrier protein ratio (w/w)for the serotype 33F conjugate is from about 1.0 to about 2.0, fromabout 1.25 to about 1.75, or from about 1.3 to about 1.7. In otherembodiments, the saccharide to carrier protein ratio (w/w) for serotype33F is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8.

In specific embodiments, the saccharide to carrier protein ratio (w/w)for the serotype 35B conjugate is from about 1.25 to about 2.25, fromabout 1.25 to about 2.0, or from about 1.3 to about 1.8. In otherembodiments, the saccharide to carrier protein ratio (w/w) for serotype33B is about 1.2, 1.3, 1.3, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0.

In specific embodiments, the saccharide to carrier protein ratio (w/w)for the serotype 24F conjugate is from about 0.5 to about 1.5, fromabout 0.75 to about 1.25, or from about 0.8 to about 1.0. In otherembodiments, the saccharide to carrier protein ratio (w/w) for serotype24F is about 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.

In a preferred embodiment, the glycoconjugate comprises less than about50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% of free polysaccharide comparedto the total amount of polysaccharide. In a preferred embodiment theglycoconjugate comprises less than about 25% of free polysaccharidecompared to the total amount of polysaccharide. In a preferredembodiment the glycoconjugate comprises less than about 20% of freepolysaccharide compared to the total amount of polysaccharide. In apreferred embodiment the glycoconjugate comprises less than about 15% offree polysaccharide compared to the total amount of polysaccharide.

IV. Methods of Use

Embodiments of the invention also include one or more of the multivalentimmunogenic compositions described herein (i) for use in, (ii) for useas a medicament or composition for, or (iii) for use in the preparationof a medicament for: (a) therapy (e.g., of the human body); (b)medicine; (c) inhibition of infection with Streptococcus pneumoniae; (d)induction of an immune response or a protective immune response againstS. pneumoniae; (e) prophylaxis of infection by S. pneumoniae; (f)prevention of recurrence of S. pneumoniae infection; (g) reduction ofthe progression, onset or severity of pathological symptoms associatedwith S. pneumoniae infection including the prevention of associatedcomplications such as brain damage, hearing loss, and seizures, (h)reduction of the likelihood of a S. pneumoniae infection or, (i)treatment, prophylaxis of, or delay in the onset, severity, orprogression of pneumococcal disease(s), including, but not limited to:pneumococcal pneumonia, pneumococcal bacteremia, pneumococcalmeningitis, otitis media and sinusitis. In these uses, the multivalentpneumococcal polysaccharide-conjugate compositions of the invention canoptionally be employed in combination with one or more adjuvants, orwithout an adjuvant.

Accordingly, the invention provides methods for the prophylactictreatment of (i.e. protection against) S. pneumoniae infection orpneumococcal disease comprising administering one or more of themultivalent immunogenic pneumococcal polysaccharide-protein conjugatecompositions of the invention to a patient in need of treatment.

The compositions and formulations of the present invention can be usedto protect or treat a human susceptible to infection, e.g., apneumococcal infection, by means of administering a composition of theinvention via a systemic or mucosal route.

In one embodiment, the invention provides a method of inducing an immuneresponse to S. pneumoniae, comprising administering to a patient animmunologically effective amount of a multivalent immunogeniccomposition of the invention. In another embodiment, the inventionprovides a method of vaccinating a human against a pneumococcalinfection, comprising the step of administering to the human animmunogically effective amount of a multivalent immunogenic compositionof the invention.

Thus, in one aspect, the invention provides a method for (1) inducing animmune response in a human patient, (2) inducing a protective immuneresponse in a human patient, (3) vaccinating a human patient against aninfection with S. pneumoniae, or (4) reducing the likelihood of a S.pneumoniae infection in a human patient, the method comprisingadministering a multivalent immunogenic composition of the invention tothe patient (i.e. any multivalent immunogenic composition describedherein, such as the multivalent immunogenic compositions described inSection II, entitled “Multivalent Immunogenic Compositions,” supra).

In one embodiment, the invention provides a method for the prevention ofpneumococcal pneumoniae and invasive disease in adults 18 years of ageand older. In another embodiment, the invention provides a method forthe prevention of pneumococcal pneumoniae and invasive disease caused bythe 24 Streptococcus pneumoniae strains (3, 6A, 6C, 7F, 8, 9N, 10A, 11A,12F, 15A, 15B, 15C, 16F, 17F, 19A, 20A, 20B, 22F, 23A, 23B, 24F, 31,33F, and 35B).

In one embodiment of the methods above, the composition comprisesmultiple S. pneumoniae polysaccharide protein conjugates wherein each ofthe conjugates comprises a polysaccharide from an S. pneumoniae serotypeconjugated to a carrier protein, wherein the serotypes of S. pneumoniaecomprise the set of serotypes: 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B,24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A. In one embodimentof the methods above, the composition comprises multiple S. pneumoniaepolysaccharide protein conjugates wherein each of the conjugatescomprises a polysaccharide from an S. pneumoniae serotype conjugated toa carrier protein, wherein the serotypes of S. pneumoniae comprise theset of serotypes: 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31,35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20. In one embodiment of themethods above, the composition comprises multiple S. pneumoniaepolysaccharide protein conjugates wherein each of the conjugatescomprises a polysaccharide from an S. pneumoniae serotype conjugated toa carrier protein, wherein the serotypes of S. pneumoniae comprise theset of serotypes: 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31,35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20B. In another embodiment ofthe methods above, the composition further comprises a S. pneumoniaepolysaccharide protein conjugate of serotype 6A or 6C.

It has been shown that a pneumococcal conjugate vaccine comprisingserotype 6A may provide some cross-protection against serotype 6C(Cooper et al., Vaccine 29 (2011) 7207-7211). Therefore, in someembodiments of the methods above, the invention also provides use ofmultivalent immunogenic compositions that do not comprise serotype 6C,but instead comprise serotype 6A or serotypes 6A and 6B. In otherembodiments, the immunogenic composition comprises pneumococcalconjugates of serotypes 6A, 6B, and 6C. In particular embodiments of themethods above, the serotypes of S. pneumoniae comprise a set ofserotypes selected from the group consisting of:

-   -   I-a) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8,        9N, 10A, 11A, 12F, 15C, 17F, and 20A;    -   I-b) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A;    -   I-c) 3, 7F, 19A, 22F, 33F, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31,        35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A; and    -   I-d) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A.        In particular embodiments of the set of serotypes above (I-a to        I-d), serotype 20 or 20B may be substituted for serotype 20A.

In further embodiments of the methods above, the serotypes of S.pneumoniae comprise a set of serotypes selected from the groupconsisting of:

-   -   II-a) 15A, 16F, 23A, 23B, 24F, 31 and 35B;    -   II-b) 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B;    -   II-c) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B;    -   II-d) 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B;    -   II-e) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F,        15C, 17F, and 20A;    -   II-f) 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A,        12F, 15C, 17F, and 20A;    -   II-g) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A,        12F, 15C, 17F, and 20A; and    -   II-h) 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A,        12F, 15C, 17F, and 20A.        In particular embodiments of the set of serotypes above (II-e to        IIh), serotype 20 or 20B may be substituted for serotype 20A.

In further embodiments of the methods above, the serotypes of S.pneumoniae comprise a set of serotypes as set forth in any ofembodiments I-a), II-a) or II-e) and further comprise serotypes 6A, 6B,and 6C.

It has also been shown that a pneumococcal conjugate vaccine comprisingserotype 10A may provide some cross-protection against serotype 39 (seeWO 2017/085586). Therefore, in some embodiments of the methods above,the invention also provides use of multivalent immunogenic compositionsthat do not comprise serotype 10A, but instead comprise serotype 39. Inother embodiments, the immunogenic composition comprises pneumococcalconjugates of serotypes 10A and 39. In particular embodiments of themethods above, the serotypes of S. pneumoniae comprise a set ofserotypes selected from the group consisting of:

-   -   III-a) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 39, 11A, 12F, 15C, 17F, and 20A;    -   III-b) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31,        35B, 8, 9N, 39, 11A, 12F, 15C, 17F, and 20A;    -   III-c) 3, 7F, 19A, 22F, 33F, 6A, 6B, 15A, 16F, 23A, 23B, 24F,        31, 35B, 8, 9N, 39, 11A, 12F, 15C, 17F, and 20A;    -   III-d) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31,        35B, 8, 9N, 39, 11A, 12F, 15C, 17F, and 20A;    -   III-e) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 39, 11A, 12F,        15C, 17F, and 20A;    -   III-f) 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 39, 11A,        12F, 15C, 17F, and 20A;    -   III-g) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 39, 11A,        12F, 15C, 17F, and 20A; and    -   III-h) 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 39, 11A,        12F, 15C, 17F, and 20A.        In particular embodiments of the set of serotypes above (II-a to        III-h), serotype 20 or 20B may be substituted for serotype 20A.

In further embodiments of the methods above, the serotypes of S.pneumoniae comprise a set of serotypes as set forth in any ofembodiments III-a) to III-h) and further comprise serotype 10A.

It has also been shown that immunogenic conjugates comprising S.pneumoniae serotype 15B capsular polysaccharide covalently linked to acarrier protein may provide some cross-protection against serotype 15Cand/or serotype 15A (see WO 2015/110942). Therefore, in some embodimentsof the methods above, the invention also provides use of multivalentimmunogenic compositions that do not comprise serotype 15C (orde-O-acetylated 15B), but instead comprise serotype 15B (i.e. theserotype 15B polysaccharide is not de-O-acetylated). In otherembodiments, the immunogenic composition comprises pneumococcalconjugates of serotypes 15B and 15C (or de-O-acetylated 15B). Inparticular embodiments of the methods above, the serotypes of S.pneumoniae comprise a set of serotypes selected from the groupconsisting of:

-   -   IV-a) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8,        9N, 10A, 11A, 12F, 15B, 17F, and 20A;    -   IV-b) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31,        35B, 8, 9N, 10A, 11A, 12F, 15B, 17F, and 20A;    -   IV-c) 3, 7F, 19A, 22F, 33F, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31,        35B, 8, 9N, 10A, 11A, 12F, 15B, 17F, and 20A;    -   IV-d) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31,        35B, 8, 9N, 10A, 11A, 12F, 15B, 17F, and 20A;    -   IV-e) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F,        15B, 17F, and 20A;    -   IV-f) 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A,        12F, 15B, 17F, and 20A;    -   IV-g) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A,        12F, 15B, 17F, and 20A; and    -   IV-h) 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A,        12F, 15B, 17F, and 20A.        In particular embodiments of the set of serotypes above (IVa to        IVh), serotype 20 or 20B may be substituted for serotype 20A.

In further embodiments of the methods above, the serotypes of S.pneumoniae comprise a set of serotypes as set forth in any ofembodiments IV-a) to IV-h) and further comprise serotype 15C (and/orde-O-acetylated 15B).

The compositions of the invention are useful in methods for providingcomplementary protection against S. pneumoniae in patients who hadpreviously received a multivalent pneumococcal vaccine. In this use, thecompositions of the invention can provide protection against particularS. pneumoniae serotypes that a patient had not been previouslyvaccinated against, can provide additional protection against S.pneumoniae serotypes that a patient had been previously vaccinatedagainst, or can provide protection against both S. pneumoniae serotypesthat a patient had not been previously vaccinated against and S.pneumoniae serotypes that a patient had been previously vaccinatedagainst.

Thus, the invention provides a method of inducing an immune response,vaccinating, or inducing a protective immune response against S.pneumoniae in a patient, comprising administering a multivalentimmunogenic composition to the patient, the composition comprisingmultiple S. pneumoniae polysaccharide protein conjugates, wherein thepolysaccharide protein conjugates comprise capsular polysaccharide froma S. pneumoniae serotype conjugated to a carrier protein, wherein thepatient had previously been vaccinated against S. pneumoniae. Inembodiments of this aspect of the invention, the multivalent immunogeniccomposition can be any multivalent immunogenic composition describedherein. In particular embodiments of the methods of the invention, themultivalent immunogenic composition is administered to a patient who waspreviously treated with a multivalent pneumococcal vaccine. Themultivalent immunogenic vaccine may be any vaccine that is indicated forthe prevention of pneumococcal disease caused by more than one serotypeof S. pneumoniae.

In specific embodiments of the method above, the patient was previouslytreated with a multivalent pneumococcal vaccine that is indicated forthe prevention of pneumococcal disease caused by S. pneumoniae serotypesselected from the group consisting of:

-   -   a) 4, 6B, 9V, 14, 18C, 19F and 23F;    -   b) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, and 19A;    -   c) 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F;    -   d) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, and        33F;    -   e) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2,        8, 9N, 10A, 11A, 12F, 15B, 17F, and 20; and    -   f) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, 33F,        8, 10A, 11A, 12F and 15B.

In specific embodiments of the method above, the multivalentpneumococcal vaccine comprises capsular polysaccharides of S. pneumoniaeserotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2,8, 9N, 10A, 11A, 12F, 15B, 17F, and 20A. In specific embodiments of themethod above, the multivalent pneumococcal vaccine comprises capsularpolysaccharides of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, 23F,1, 3, 5, 7F, 19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, and 20.In specific embodiments of the method above, the multivalentpneumococcal vaccine comprises capsular polysaccharides of S. pneumoniaeserotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2,8, 9N, 10A, 11A, 12F, 15B, 17F, and 20B.

In specific embodiments of the method above, the multivalentpneumococcal vaccine comprises multiple polysaccharide proteinconjugates, wherein the polysaccharide protein conjugates comprisepolysaccharide from a S. pneumoniae serotype conjugated to a carrierprotein. In other embodiments, the multivalent pneumococcal vaccinecomprises multiple S. pneumoniae capsular polysaccharides that are notconjugated to a carrier protein.

In additional embodiments of the method above, the patient waspreviously treated with PREVNAR® 13 (Pneumococcal 13-valent ConjugateVaccine [Diphtheria CRM197 Protein], Pfizer, Inc., Philadelphia, PA,USA).

In further embodiments of the method above, the patient was previouslytreated with PNEUMOVAX® 23 (Pneumococcal Vaccine Polyvalent, Merck &Co., Inc., Kenilworth, NJ, USA).

In still further embodiments of the method above, the patient waspreviously treated with SYNFLORIX™ (Pneumococcal polysaccharideconjugate vaccine (adsorbed), GlaxoSmithKline Biologicals s.a.,Rixensart, Belgium).

In embodiments of the method above, the multivalent immunogeniccomposition of the invention is administered to a patient at any timeafter the patient has received a multivalent pneumococcal vaccine,according to the treatment regimen provided by the medical professional,e.g. a physician. In particular embodiments, the multivalent immunogeniccomposition of the invention is administered to a patient from 1 monthto 5 years after the patient has received the multivalent pneumococcalvaccine, alternatively, from 1 month to 1 year, from 1 month to 2 years,from 1 month to 3 years, from 1 month to 4 years, from 1 month to 6months, from 2 months to 6 months, from 2 months to 1 year, from 1 yearto 5 years, from 6 months to 5 years, from 6 months to 4 years, from 6months to 3 years, from 6 months to 2 years, from 6 months to 1 year,from 1 year to 4 years, from 1 year to 3 years, or from 1 year to 2years, after the patient has received the multivalent pneumococcalvaccine. In further embodiments, the multivalent immunogenic compositionis administered to the patient about 1 month, about 2 months, about 3months, about 4 months, about 5 months, about 6 months, about 7 months,about 8 months, about 9 months, about 10 months, about 11 months, about1 year, about 1.25 years, about 1.5 years, about 1.75 years, about 2years, about 2.25 years, about 2.5 years, about 2.75 years, about 3years, about 3.25 years, about 3.5 years, about 3.75 years, about 4years, about 4.25 years, about 4.5 years, about 4.75 years, or about 5years after the patient has received the multivalent pneumococcalvaccine.

In further embodiments, the invention provides a method for (1) inducingan immune response in a human patient, (2) inducing a protective immuneresponse in a human patient, (3) vaccinating a human patient against aninfection with S. pneumoniae, or (4) reducing the likelihood of a S.pneumoniae infection in a human patient, the method comprisingadministering a multivalent immunogenic composition of the invention andadministering a multivalent pneumococcal vaccine to the patient, in anyorder. The multivalent pneumococcal vaccine may be any vaccine indicatedfor the prevention of pneumococcal disease caused by more than oneserotype of S. pneumoniae.

In specific embodiments of the method above, the patient is treated witha multivalent immunogenic composition of the invention and a multivalentpneumococcal vaccine that is indicated for the prevention ofpneumococcal disease caused by S. pneumoniae serotypes selected from thegroup consisting of:

-   -   a) 4, 6B, 9V, 14, 18C, 19F and 23F;    -   b) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, and 19A;    -   c) 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F;    -   d) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, and        33F;    -   e) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2,        8, 9N, 10A, 11A, 12F, 15B, 17F, and 20; and    -   f) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, 33F,        8, 10A, 11A, 12F and 15B.

In specific embodiments of the method above, the multivalentpneumococcal vaccine comprises capsular polysaccharides of S. pneumoniaeserotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2,8, 9N, 10A, 11A, 12F, 15B, 17F, and 20A.

In specific embodiments of the method above, the multivalentpneumococcal vaccine comprises multiple polysaccharide proteinconjugates, wherein the polysaccharide protein conjugates comprisepolysaccharide from a S. pneumoniae serotype conjugated to a carrierprotein. In other embodiments, the multivalent pneumococcal vaccinecomprises multiple S. pneumoniae capsular polysaccharides that are notconjugated to a carrier protein.

In additional embodiments of the method above, the patient is treatedwith a multivalent immunogenic composition of the invention and istreated with PREVNAR® 13 (Pneumococcal 13-valent Conjugate Vaccine[Diphtheria CRM197 Protein], Pfizer, Inc., Philadelphia, PA, USA), inany order. In one embodiment, the patient is administered PREVNAR® 13first and the patient is administered a multivalent immunogeniccomposition of the invention second. In alternative embodiments, thepatient is administered a multivalent immunogenic composition of theinvention first and is administered PREVNAR® 13 second.

In further embodiments of the method above, the patient is treated witha multivalent immunogenic composition of the invention and is treatedwith PNEUMOVAX® 23 (pneumococcal vaccine polyvalent, Merck & Co., Inc.,Kenilworth, NJ, USA), in any order. In one embodiment, the patient isadministered PNEUMOVAX® 23 first and the patient is administered amultivalent immunogenic composition of the invention second. Inalternative embodiments, the patient is administered a multivalentimmunogenic composition of the invention first and is administeredPNEUMOVAX® 23 second.

In still further embodiments of the method above, the patient is treatedwith a multivalent immunogenic composition of the invention and istreated with SYNFLORIX™ (Pneumococcal polysaccharide conjugate vaccine(adsorbed), GlaxoSmithKline Biologicals s.a., Rixensart, Belgium), inany order. In one embodiment, the patient is administered SYNFLORIX™first and the patient is administered a multivalent immunogeniccomposition of the invention second. In alternative embodiment, thepatient is administered a multivalent immunogenic composition of theinvention first and is administered SYNFLORIX™ second.

In some embodiments of the method above, the multivalent immunogeniccomposition and the multivalent pneumococcal vaccine are administeredconcurrently. As used herein, “concurrent administration” is not limitedto dosing of two compositions at the same time, but includesadministration one right after the other in any order. In someembodiments, the multivalent immunogenic composition and the multivalentpneumococcal vaccine are administered via intramuscular or subcutaneousadministration into separate anatomical sites, e.g. two different arms.

In some embodiments of the method above, the amount of time betweenadministration of the multivalent immunogenic composition of theinvention and the multivalent pneumococcal vaccine is from about 4 weeksto about 1 year. In alternative embodiments, the amount of time is fromabout 1 month to about 5 years.

In one embodiment, the patient is administered the multivalentpneumococcal vaccine first and the multivalent immunogenic compositionof the invention second. In alternative embodiments, the patient isadministered a multivalent immunogenic composition of the inventionfirst and is administered the multivalent pneumococcal vaccine second.

Also provided is a method of inducing an immune response, vaccinating orinducing a protective immune response against S. pneumoniae in apatient, comprising:

-   -   (1) administering a multivalent immunogenic composition to the        patient, the composition comprising multiple S. pneumoniae        polysaccharide protein conjugates, wherein each of the        polysaccharide protein conjugates comprise capsular        polysaccharide from a S. pneumoniae serotype conjugated to a        carrier protein,    -   (2) waiting for a pre-determined amount of time to pass, and    -   (3) administering a multivalent pneumococcal vaccine to the        patient. In this method, the multivalent immunogenic composition        can comprise any combination of S. pneumoniae polysaccharide        protein conjugates set forth herein and the multivalent        pneumococcal vaccine can be any vaccine indicated for the        prevention of disease caused by more than one serotype of S.        pneumoniae.

Also provided by the invention is a method of inducing an immuneresponse, vaccinating or inducing a protective immune response againstS. pneumoniae in a patient, comprising:

-   -   (1) administering a multivalent pneumococcal vaccine to the        patient,    -   (2) waiting for a pre-determined amount of time to pass, and    -   (3) administering a multivalent immunogenic composition to the        patient, the composition comprising multiple S. pneumoniae        polysaccharide protein conjugates, wherein each of the        polysaccharide protein conjugates comprise capsular        polysaccharide from a S. pneumoniae serotype conjugated to a        carrier protein.

In this method, the multivalent immunogenic composition can comprise anycombination of S. pneumoniae polysaccharide protein conjugates set forthherein and the multivalent pneumococcal vaccine can be any vaccineindicated for the prevention of disease caused by more than one serotypeof S. pneumoniae.

In some embodiments of the methods above, the multivalent pneumococcalvaccine comprises multiple S. pneumoniae polysaccharide proteinconjugates, wherein the polysaccharide protein conjugates comprisecapsular polysaccharide from a S. pneumoniae serotype conjugated to acarrier protein. In alternative embodiments, the multivalentpneumococcal vaccine comprises S. pneumoniae capsular polysaccharidesthat are not conjugated to a carrier protein.

In any embodiments of the methods of the invention (i.e. any of themethods described herein), the method may further comprise administeringone or more additional doses of a multivalent immunogenic composition ofthe invention to the patient. In such methods, the patient may havealready received a multivalent pneumococcal vaccine prior to receiving afirst dose of a multivalent immunogenic composition of the invention,supra, or may not have been vaccinated against S. pneumoniae prior toreceiving a multivalent immunogenic composition of the invention. Thus,in one embodiment, a patient who had received a multivalent pneumococcalvaccine indicated for the prevention of pneumococcal disease caused byS. pneumoniae is administered two or more doses of a multivalentimmunogenic composition of the invention. In alternative embodiments, apatient who had not been previously treated with any vaccine indicatedfor the prevention of pneumococcal disease, is administered two or moredoses of a multivalent immunogenic composition of the invention.

In embodiments of the method above, the two or more doses are of thesame multivalent immunogenic composition of the invention. Inalternative embodiments, the two or more doses are of differentmultivalent immunogenic compositions of the invention.

In specific embodiments of any of these methods, the patient isadministered two, three, or four doses of a multivalent immunogeniccomposition of the invention. In particular embodiments, the patient isimmunocompromised (e.g., on an immunosuppressive regimen following astem cell transplant) and the number of doses is three.

In some embodiments, the amount of time between administration of eachdose of multivalent immunogenic composition of the invention is fromabout 4 weeks to about 1 year. In alternative embodiment, the amount oftime between administration of each dose of multivalent immunogeniccomposition of the invention is from about 1 month to about 5 years.

In embodiments of any of the methods of the invention, the patient to betreated with the composition(s) of the invention is a human. In certainembodiments, the human patient is a toddler (approximately 12 to 24months), or young child (approximately 2 to 5 years). The compositionsof this invention are also suitable for use with older children,adolescents and adults (e.g., aged 18 to 45 years, aged 18 to 50 years,aged 18 to 55 years, aged 18 to 60 years or 18 to 65 years). In otherembodiments of any of the methods of the invention, the patient is fromabout 2 to about 18 years of age. In further embodiments of any of themethods of the invention, the patient is 18 years of age or older.

In further embodiments of the methods of the invention, the humanpatient is elderly. In some embodiments of any of the methods of theinvention, the patient is 50 years of age or older. In some embodimentsof any of the methods of the invention, the patient is 55 years of ageor older. In some embodiments of any of the methods of the invention,the patient is 60 years of age or older. In still further embodiments ofany of the methods of the invention, the patient is 65 years of age orolder. In additional embodiments of any of the methods of the invention,the patient is 70 years of age or older.

In some embodiments of any of the methods of the invention, the patientto be treated with an immunogenic composition of the invention isimmunocompromised.

In some embodiments of any of the methods of the invention, themultivalent immunogenic composition is administered concomitantly with avaccine against influenza. In certain embodiments, the influenza vaccineis a “senior flu vaccine,” a high dose flu vaccine indicated for theelderly, e.g. persons aged 65 and older.

The invention provides a method for inducing a protective immuneresponse in a patient against a pneumococcal infection comprising thestep of administering to the patient an immunologically effective amountof any of the multivalent immunogenic pneumococcalpolysaccharide-protein conjugate compositions described herein. Optimalamounts of components for a particular vaccine (i.e. multivalentimmunogenic composition) can be ascertained by standard studiesinvolving observation of appropriate immune responses in subjects. Forexample, in another embodiment, the dosage for human vaccination isdetermined by extrapolation from animal studies to human data. Inanother embodiment, the dosage is determined empirically.

The methods of the invention can be used for the prevention and/orreduction of primary clinical syndromes caused by microbes, e.g., S.pneumonia, including both invasive infections (meningitis, pneumonia,and bacteremia), and noninvasive infections (acute otitis media, andsinusitis).

Administration of the compositions of the invention can include one ormore of injection via the intramuscular, intraperitoneal, intradermal orsubcutaneous routes; or via mucosal administration to theoral/alimentary, respiratory or genitourinary tracts. In one embodiment,intranasal administration is used for the treatment of pneumonia orotitis media (as nasopharyngeal carriage of pneumococci can be moreeffectively prevented, thus attenuating infection at its earlieststage). In specific embodiments, the compositions of the invention areadministered to the patient via intramuscular or subcutaneousadministration.

Additional Embodiments of the Instant Invention

(1) A multivalent immunogenic composition comprising S. pneumoniaepolysaccharide protein conjugates wherein each of the conjugatescomprises a polysaccharide from an S. pneumoniae serotype conjugated toa carrier protein, wherein the serotypes of S. pneumoniae in thepolysaccharide protein conjugates comprise a set of serotypes selectedfrom the group consisting of:

-   -   a) 15A, 16F, 23A, 23B, 24F, 31 and 35B;    -   b) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C,        17F, and 20; and    -   c) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8,        9N, 10A, 11A, 12F, 15C, 17F, and 20.

(2) The multivalent immunogenic composition of embodiment 1, wherein theset of serotypes of S. pneumoniae listed in a), b) or c) furthercomprises:

-   -   (i) serotype 6C,    -   (ii) serotype 6A, or    -   (iii) serotypes 6A and 6B.

(3) The multivalent immunogenic composition of embodiment 1, wherein theserotypes of S. pneumoniae comprise a set of serotypes selected from thegroup consisting of:

-   -   a) 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F,        15C, 17F, and 20;    -   b) 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F,        15C, 17F, and 20; and    -   c) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A,        12F, 15C, 17F, and 20        wherein serotype 20 in sets a) to c) may be optionally        substituted with either serotype 20A or serotype 20B.

(4) The multivalent immunogenic composition of embodiment 1, wherein theserotypes of S. pneumoniae comprise a set of serotypes selected from thegroup consisting of:

-   -   a) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A;    -   b) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A; and    -   c) 3, 7F, 19A, 22F, 33F, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31,        35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A.

(5) The multivalent immunogenic composition of embodiment 1 wherein theserotypes of S. pneumoniae comprise serotypes selected from the groupconsisting of 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31,35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A.

(6) The multivalent immunogenic composition of embodiment 1 wherein theserotypes of S. pneumoniae comprise serotypes selected from the groupconsisting of 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31,35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A.

(7) The multivalent immunogenic composition of any of embodiments 1 to6, wherein the immunogenic composition does not comprise any further S.pneumoniae polysaccharide protein conjugates.

(8) The multivalent immunogenic composition of any of embodiments 1 to7, wherein at least one of the polysaccharide protein conjugates isformed by a conjugation reaction comprising an aprotic solvent.

(9) The multivalent immunogenic composition of any of embodiments 1 to8, wherein each of the polysaccharide protein conjugates is formed by aconjugation reaction comprising an aprotic solvent.

(10) The multivalent immunogenic composition of embodiments 1 to 9,wherein the total polysaccharide concentration in the composition isfrom about 0.02 to about 0.175 mg/mL

(11) The multivalent immunogenic composition of any of embodiments 1 to10, wherein the weight average molecular weight (Mw) of the S.pneumoniae polysaccharide protein conjugates in the composition is fromabout 1,000 to about 6,000 kDa.

(12) The multivalent immunogenic composition of any of embodiments 1 to11, wherein the number average molecular weight (Mn) of the S.pneumoniae polysaccharide protein conjugates in the composition is fromabout 500 to about 4,000 kDa.

(13) The multivalent immunogenic composition of any of embodiments 1 to12, wherein the composition is stable for up to 4 weeks or more at 37°C.

(14) The multivalent immunogenic composition of any of embodiments 1 to12, wherein the composition is stable for up to 12 weeks or more at 4°C.

(15) The multivalent immunogenic composition of embodiment 13 or 14,wherein stability is determined using HPSEC UV/MALS/RI.

(16) The multivalent immunogenic composition of any of embodiments 1 to15, wherein the emission maximum of the composition measured usingintrinsic protein fluorescence spectroscopy with an excitationwavelength at 280 nanometers (nm) is from about 335 nm to about 342 nm.

(17) The multivalent immunogenic composition of embodiment 16, whereinthe emission maximum remains from about 335 nm to about 342 nm and thefluorescence intensity is stable for at least 1 week at 37° C.

(18) The multivalent immunogenic composition of any of embodiments 8-17,wherein the aprotic solvent is dimethylsulfoxide (DMSO).

(19) The multivalent immunogenic composition of embodiment 8 or 9,wherein the conjugation reaction is reductive amination.

(20) The multivalent immunogenic composition of any one of embodiments 1to 19, wherein the carrier protein is selected from the group consistingof Outer Membrane Protein Complex (OMPC), tetanus toxoid, diphtheriatoxoid, protein D and CRM197.

(21) The multivalent immunogenic composition of any one of embodiments 1to 20, wherein the carrier protein is CRM197.

(22) The multivalent immunogenic composition of any one of embodiments 1to 21, wherein the composition further comprises an adjuvant.

(23) The multivalent immunogenic composition of any one of embodiments 1to 21, wherein the composition does not comprise an adjuvant.

(24) The multivalent immunogenic composition of any one of embodiments 1to 23, further comprising 10-80 mM histidine pH 5.8 and 150 mM NaCl.

(25) The multivalent immunogenic composition of embodiment 24, furthercomprising from 0.025% to 0.8% w/v polysorbate 20.

(26) A multivalent immunogenic composition comprising S. pneumoniaepolysaccharide protein conjugates, wherein each of the conjugatescomprises a polysaccharide from an S. pneumoniae serotype conjugated toCRM197, wherein the serotypes of S. pneumoniae in the polysaccharideprotein conjugates consist of a set of serotypes selected from the groupconsisting of:

-   -   a) 15A, 16F, 23A, 23B, 24F, 31 and 35B;    -   b) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B;    -   c) 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B;    -   d) 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B;    -   e) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C,        17F, and 20A;    -   f) 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F,        15C, 17F, and 20A;    -   g) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A,        12F, 15C, 17F, and 20A;    -   h) 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F,        15C, 17F, and 20A;    -   i) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8,        9N, 10A, 11A, 12F, 15C, 17F, and 20A;    -   j) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A;    -   k) 3, 7F, 19A, 22F, 33F, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31,        35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A; and    -   l) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A;        wherein serotype 20A in sets e) to 1) may be optionally        substituted with either serotype 20 or serotype 20B; wherein        each of the polysaccharide protein conjugates is formed by a        conjugation reaction comprising an aprotic solvent and wherein        the composition does not comprise an adjuvant.

(27) A multivalent immunogenic composition of embodiment 26 comprisingS. pneumoniae polysaccharide protein conjugates, wherein each of theconjugates comprises a polysaccharide from an S. pneumoniae serotypeconjugated to CRM197, wherein the serotypes of S. pneumoniae in thepolysaccharide protein conjugates consist of serotypes selected from 3,7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A,11A, 12F, 15C, 17F, and 20A; wherein serotype 20A may be optionallysubstituted with either serotype 20 or serotype 20B; wherein each of thepolysaccharide protein conjugates is formed by a conjugation reactioncomprising an aprotic solvent and wherein the composition does notcomprise an adjuvant.

(28) A multivalent immunogenic composition of embodiment 26 comprisingS. pneumoniae polysaccharide protein conjugates, wherein each of theconjugates comprises a polysaccharide from an S. pneumoniae serotypeconjugated to CRM197, wherein the serotypes of S. pneumoniae in thepolysaccharide protein conjugates consist of serotypes selected from 3,7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A,11A, 12F, 15C, 17F, and 20A; wherein serotype 20A may be optionallysubstituted with either serotype 20 or serotype 20B; wherein each of thepolysaccharide protein conjugates is formed by a conjugation reactioncomprising an aprotic solvent and wherein the composition does notcomprise an adjuvant.

(29) A method for inducing a protective immune response in a humanpatient comprising administering the multivalent immunogenic compositionof any one of embodiments 1 to 28 to the patient.

(30) The method of embodiment 29, wherein the serotypes of S. pneumoniaecomprise a set of serotypes selected from the group consisting of:

-   -   a) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8,        9N, 10A, 11A, 12F, 15C, 17F, and 20A;    -   b) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A;    -   c) 3, 7F, 19A, 22F, 33F, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31,        35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A; and    -   d) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A;        wherein serotype 20A in sets a) to d) may be optionally        substituted with either serotype 20 or serotype 20B.

(31) The method of embodiment 29, wherein the serotypes of S. pneumoniaecomprise a set of serotypes selected from the group consisting of:

-   -   a) 15A, 16F, 23A, 23B, 24F, 31 and 35B;    -   b) 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B 6A, 6B, 15A, 16F,        23A, 23B, 24F, 31 and 35B;    -   c) 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B;    -   d) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C,        17F, and 20A;    -   e) 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F,        15C, 17F, and 20A;    -   f) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A,        12F, 15C, 17F, and 20A; and    -   g) 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F,        15C, 17F, and 20A;        wherein serotype 20A in sets d) to g) may be optionally        substituted with either serotype 20 or serotype 20B.

(32) The method of embodiment 30, wherein the serotypes of S. pneumoniaecomprise serotypes selected from 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F,23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A.

(33) The method of embodiment 30, wherein the serotypes of S. pneumoniaecomprise serotypes selected from 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F,23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A.

(34) The method of any of embodiments 29 to 33, wherein the patient waspreviously treated with a multivalent pneumococcal vaccine.

(35) The method of embodiment 34, wherein the multivalent pneumococcalvaccine is indicated for the prevention of pneumococcal disease causedby serotypes selected from the group consisting of:

-   -   a) 4, 6B, 9V, 14, 18C, 19F and 23F;    -   b) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, and 19A;    -   c) 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F;    -   d) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, and        33F;    -   e) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2,        8, 9N, 10A, 11A, 12F, 15B, 17F, and 20;    -   f) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, 33F,        8, 10A, 11A, 12F and 15B; and    -   g) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 6C, 7F, 19A, 22F,        33F, 8, 10A, 11A, 12F and 15B

(36) The method of embodiment 34 or 35, wherein the multivalentpneumococcal vaccine comprises multiple polysaccharide proteinconjugates, wherein the polysaccharide protein conjugates comprisepolysaccharide from a S. pneumoniae serotype conjugated to a carrierprotein.

(37) The method of any of embodiments 31 to 36, further comprisingadministering one or more additional doses of the multivalentimmunogenic composition to the patient.

(38) The method of embodiment 37, wherein the amount of time betweendoses is from about 4 weeks to about 1 year.

(39) The method of embodiment 37 or claim 38, wherein two additionaldoses are administered to the patient and the patient isimmunocompromised.

(40) The method of any of embodiments 31 to 33, further comprisingadministering a multivalent pneumococcal vaccine to the patient in anyorder, wherein the multivalent immunogenic composition and themultivalent pneumococcal vaccine are not the same.

(41) The method of embodiment 40, wherein the multivalent pneumococcalvaccine is indicated for the prevention of pneumococcal disease causedby S. pneumoniae serotypes selected from the group consisting of

-   -   a) 4, 6B, 9V, 14, 18C, 19F and 23F;    -   b) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, and 19A;    -   c) 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F;    -   d) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, and        33F;    -   e) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2,        8, 9N, 10A, 11A, 12F, 15B, 17F, and 20;    -   f) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, 33F,        8, 10A, 11A, 12F and 15B; and    -   g) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 6C, 7F, 19A, 22F,        33F, 8, 10A, 11A, 12F and 15B.

(42) The method of embodiment 41, wherein the multivalent pneumococcalvaccine is comprised of multiple S. pneumoniae polysaccharide proteinconjugates wherein each of the conjugates comprises polysaccharide froman S. pneumoniae serotype conjugated to a carrier protein.

(43) The method of any of embodiments 40-42, wherein the multivalentimmunogenic composition and the multivalent pneumococcal vaccine areadministered concurrently.

(44) The method of any of embodiments 40-42, wherein the amount of timebetween administration of the multivalent immunogenic composition andthe multivalent pneumococcal vaccine is from about 4 weeks to about 1year.

(45) The method of any of embodiments 40-42 or 44, wherein themultivalent immunogenic composition is administered before themultivalent pneumococcal vaccine.

(46) The method of any of embodiments 40-42 or 44, wherein themultivalent pneumococcal vaccine is administered before the multivalentimmunogenic composition

(47) The method of any of embodiments 31 to 46, wherein the patient is50 years of age or older.

(48) The method of any of embodiments 31 to 46, wherein the patient isbetween 2 and 18 years of age.

(49) The method of any of embodiments 31 to 46, wherein the patient is18 years of age or older.

(50) The method of any of embodiments 31 to 46, wherein the patient is65 years of age or older.

(51) The method of any of embodiments 31 to 50, wherein the patient isimmunocompromised.

(52) The method of any of embodiments 31 to 51, wherein the immunogeniccomposition is administered by subcutaneous or intramuscular injection.

(53) The method of any of embodiments 31 to 52, wherein the immunogeniccomposition is administered concomitantly with a vaccine againstinfluenza.

(54) A method for preparing a serotype 8 Streptococcus pneumoniaepolysaccharide-protein conjugate utilizing a conjugation reaction in anaprotic solvent, wherein the conjugation reaction does not usecyanoborohydride.

(55) The method of embodiment 54, wherein the conjugation reaction is aSchiff base reduction or reductive amination.

(56) The method of embodiments 54 and 55, wherein the protein is tetanustoxoid, diphtheria toxoid, or CRM197.

(57) The method of embodiment 56, wherein the protein is CRM197.

(58) The method of any one of embodiments 54 to 57, wherein theconjugation reaction is reductive amination.

(59) The method of any of embodiments 54 to 48, wherein the reductiveamination is performed in dimethylsulfoxide (DMSO).

(60) A method for inducing a protective immune response in a humanpatient comprising administering the multivalent immunogenic compositionof embodiments 1 to 28 to the human patient, wherein the polysaccharidedose of each serotype is from about 0.4 to about 4 μg.

(61) A multivalent immunogenic composition comprising S. pneumoniaepolysaccharide protein conjugates, wherein each of the conjugatescomprises a polysaccharide from a S. pneumoniae serotype conjugated to acarrier protein, and wherein the polysaccharide protein conjugatesinclude polysaccharides of S. pneumoniae serotypes selected from thegroup consisting of:

-   -   I) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20;    -   II) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A;    -   III) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20B;    -   IV) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20;    -   V) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A; and    -   VI) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B,        8, 9N, 10A, 11A, 12F, 15C, 17F, and 20B.

(62) The multivalent immunogenic composition of embodiment 61, whereinthe immunogenic composition does not comprise polysaccharide proteinconjugates having polysaccharides from any further S. pneumoniaeserotypes.

(63) The multivalent immunogenic composition of any of embodiments 61 or62, wherein at least one of the polysaccharide protein conjugates isformed by a conjugation reaction comprising an aprotic solvent.

(64) The multivalent immunogenic composition of any of embodiments 61 to63, wherein each of the polysaccharide protein conjugates is formed by aconjugation reaction comprising an aprotic solvent.

(65) The multivalent immunogenic composition of any of embodiments 63 to64, wherein the aprotic solvent is dimethylsulfoxide (DMSO).

(66) The multivalent immunogenic composition of any of embodiments 61 to65, wherein the carrier protein is selected from the group consisting ofOuter Membrane Protein Complex (OMPC), tetanus toxoid, diphtheriatoxoid, protein D and CRM197.

(67) The multivalent immunogenic composition of any of embodiments 61 to66, wherein the carrier protein is CRM197.

(68) The multivalent immunogenic composition of any of embodiments 61 to67, wherein the composition further comprises an adjuvant.

(69) The multivalent immunogenic composition of any of embodiments 61 to67, wherein the composition does not comprise an adjuvant.

(70) A method for inducing a protective immune response in a humanpatient comprising administering the multivalent immunogenic compositionof any of embodiments 61 to 69 to a patient.

(71) The method of embodiment 70, wherein the patient was previouslytreated with a multivalent pneumococcal vaccine.

(72) The method of any of embodiments 70 to 71, further comprisingadministering one or more additional doses of the multivalentimmunogenic composition to the patient.

(73) The method of any of embodiments 70 to 72, wherein the amount oftime between doses is from about 4 weeks to about 1 year.

(74) The method of any of embodiments 70 to 73, wherein two additionaldoses are administered to the patient and the patient isimmunocompromised.

(75) The method of any of embodiments 70 to 74, further comprisingadministering a multivalent pneumococcal vaccine to the patient in anyorder, wherein the multivalent immunogenic composition and themultivalent pneumococcal vaccine are not the same.

(76) The method of embodiments 75, wherein the multivalent pneumococcalvaccine is comprised of multiple S. pneumonia polysaccharide proteinconjugates wherein each of the conjugates comprises polysaccharide froma S. pneumoniae serotype conjugated to a carrier protein.

(77) The method of any of embodiments 75 to 76, wherein the multivalentimmunogenic composition and the multivalent pneumococcal vaccine areadministered concurrently.

(78) The method of any of embodiments 75 to 77, wherein the amount oftime between administration of the multivalent immunogenic compositionand the multivalent pneumococcal vaccine is from about 4 weeks to about1 year.

(79) The method of any of embodiments 75 to 78, wherein the multivalentimmunogenic composition is administered before the multivalentpneumococcal vaccine.

(80) The method of any of embodiments 75 to 78, wherein the multivalentpneumococcal vaccine is administered before the multivalent immunogeniccomposition.

(81) The method of any of embodiments 70 to 80, wherein the patient is50 years of age or older.

(82) The method of any of embodiments 70 to 81, wherein the patient isbetween 2 and 18 years of age.

(83) The method of any of embodiments 70 to 82, wherein the patient is18 years of age or older.

(84) The method of any of embodiments 70 to 83, wherein the patient is65 years of age or older.

(85) The method of any of embodiments 70 to 84, wherein the patient isimmunocompromised.

(86) The method of any of embodiments 70 to 85, wherein the immunogeniccomposition is administered by subcutaneous or intramuscular injection.

(87) The method of any of embodiments 70 to 86, wherein the immunogeniccomposition is administered concomitantly with a vaccine againstinfluenza.

(88) A method for the prevention of pneumococcal pneumoniae and invasivedisease in adults 18 years of age and older comprising administering themultivalent immunogenic composition of embodiment 62 to a patient.

(89) A method for the prevention of pneumococcal pneumoniae and invasivedisease caused by the 24 Streptococcus pneumoniae strains (3, 6A, 6C,7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 19A, 20A, 20B, 22F,23A, 23B, 24F, 31, 33F, and 35B) comprising administering themultivalent immunogenic composition of embodiment 62 to a patient.

(90) A multivalent immunogenic composition comprising 21 distinctpolysaccharide protein conjugates, wherein each of the conjugatescomprises a capsular polysaccharide from a S. pneumoniae serotypeconjugated to a carrier protein, wherein the polysaccharide are preparedfrom S. pneumoniae serotypes 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A,23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A and whereinthe carrier protein is CRM197.

(91) The multivalent immunogenic composition of embodiment 90, whereinthe immunogenic composition does not comprise polysaccharide proteinconjugates prepared from any other S. pneumoniae serotypes.

(92) The multivalent immunogenic composition of embodiment 90, whereineach of the polysaccharide protein conjugates is formed by a conjugationreaction comprising an aprotic solvent, wherein the aprotic solvent isdimethylsulfoxide (DMSO).

(93) The multivalent immunogenic composition of embodiment 90, whereinthe composition does not comprise an adjuvant.

All publications mentioned herein are incorporated by reference for thepurpose of describing and disclosing methodologies and materials thatmight be used in connection with the present invention.

Having described different embodiments of the invention herein withreference to the accompanying drawings, it is to be understood that theinvention is not limited to those precise embodiments, and that variouschanges and modifications may be effected therein by one skilled in theart without departing from the scope or spirit of the invention asdefined in the appended claims.

The following examples illustrate, but do not limit the invention.

Example 1

Preparation of S. pneumoniae Capsular Polysaccharides

Methods of culturing pneumococci are well known in the art. See, e.g.,Chase, 1967, Methods of Immunology and Immunochemistry 1:52. Methods ofpreparing pneumococcal capsular polysaccharides are also well known inthe art. See, e.g., European Patent No. EP 0 497 524 B1. The processdescribed below generally follows the method described in EuropeanPatent No. EP 0 497 524 B1 and is generally applicable to allpneumococcal serotypes.

Isolates of pneumococcal strains for serotypes 6C, 23B, and 31 wereobtained from Centers for Disease Control and Prevention (Atlanta, GA).Strains for serotypes 3, 8, 10A, 11A, 12F, 15B, 22F, and 33F wereobtained from the University of Pennsylvania (Dr. Robert Austrian).Strains for serotypes 17F and 19A were obtained from the FDA Office ofBiologics (Dr. John Robbins). Serotype 7F was obtained from the StateUniversity of New York, Downstate Medical Center (Dr. Gerald Schiffman).Isolates of pneumococcal serotypes not listed above were obtained fromthe American Type Culture Collection (Manassas, VA). Where needed,subtypes were differentiated on the basis of Quellung reaction usingspecific antisera. See, e.g., U.S. Pat. No. 5,847,112. The obtainedisolates were further clonally isolated by plating serially in twostages on agar plates consisting of an animal-component free mediumcontaining soy peptone, yeast extract, and glucose without hemin. Forserotype 7F, the agar plates used also contained hemin. Clonal isolatesfor each serotype were further expanded in liquid culture usinganimal-component free media containing soy peptone, yeast extract,HEPES, sodium chloride, sodium bicarbonate, potassium phosphate,glucose, and glycerol to prepare the pre-master cell banks.

The production of each serotype of pneumococcal polysaccharide consistedof a cell expansion and batch production fermentation followed bychemical inactivation prior to downstream purification. A thawed cellbank vial from each serotype was expanded using a shake flask or culturebottle containing a pre-sterilized animal-component free growth mediacontaining soy peptone or soy peptone ultrafiltrate, yeast extract oryeast extract ultrafiltrate, HEPES, sodium chloride, sodium bicarbonate,potassium phosphate, and glucose. The cell expansion culture was grownin a sealed shake flask or bottle to minimize gas exchange withtemperature and agitation control. For serotypes 3, 7F, 8, 9N, 10A, 11A,12F, 15B, 17F, 19A, 20, 22F, and 33F, a thawed cell bank vial wasexpanded using a fermentor containing the same media. During the cellexpansion of these serotypes, temperature, pH, pressure, and agitationwere controlled. Airflow overlay was also controlled as sparging was notused. After achieving a specified culture density, as measured byoptical density at 600 nm, a portion of the cell expansion culture wastransferred to a production fermentor containing pre-sterilizedanimal-component free growth media containing soy peptone or soy peptoneultrafiltrate, yeast extract or yeast extract ultrafiltrate, sodiumchloride, potassium phosphate, and glucose. Temperature, pH, pressure,and agitation were controlled. Airflow overlay was also controlled assparging was not used.

The batch fermentation was terminated via the addition of a chemicalinactivating agent, phenol, when glucose was nearly exhausted. Purephenol was added to a final concentration of 0.8-1.2% to inactivate thecells and liberate the capsular polysaccharide from the cell wall.Primary inactivation occurs for a specified time within the fermentorwhere temperature and agitation continue to be controlled. After primaryinactivation, the batch was transferred to another vessel where it washeld for an additional specified time at controlled temperature andagitation for complete inactivation. This was confirmed by eithermicrobial plating techniques or by verification of the phenolconcentration and specified time. The inactivated broth was thenpurified.

Example 2

Purification of Pneumococcal Polysaccharides

The purification process for the pneumococcal polysaccharides consistedof several centrifugation, depth filtration, concentration/diafiltrationoperations, and precipitation steps. All procedures were performed atroom temperature unless otherwise specified.

Inactivated broth from the fermentor cultures of S. pneumoniae wereflocculated with a cationic polymer (such as BPA-1000, TRETOLITE® (BakerHughes Inc., Houston, TX), Spectrum 8160, poly(ethyleneimine), andMillipore pDADMAC). The cationic polymers binded to the impurityproteins, nucleic acids and cell debris. Following the flocculation stepand an aging period, flocculated solids were removed via centrifugationand multiple depth filtration steps. Clarified broth was concentratedand diafiltered using a 100 kDa to 500 kDa MWCO (molecular weightcutoff) filter. Diafiltration was accomplished using Tris, MgCl₂ bufferand sodium phosphate buffer. Diafiltration removed residual nucleic acidand protein.

Removal of further impurities was accomplished by reprecipitation of thepolysaccharide in sodium acetate and phenol with denatured alcoholand/or isopropanol. During the phenol precipitation step, sodium acetatein sodium phosphate saline buffer and phenol (liquefied phenols or solidphenols) were charged to the diafiltered retentate. Alcoholfractionation of the polysaccharide was then conducted in two stages. Inthe first stage a low percent alcohol was added to the preparation toprecipitate cellular debris and other unwanted impurities, while thecrude polysaccharide remained in solution. The impurities were removedvia centrifugation followed by a depth filtration step. Thepolysaccharide was then recovered from the solution by adding additionalisopropanol or denatured alcohol to the batch. The precipitatedpolysaccharide pellet was recovered by centrifugation, triturated anddried as a powder and stored frozen at −70° C.

Example 3

Structure Identity Analysis of Certain Pneumococcal Serotypes by NMRTesting

Samples for NMR analysis were prepared by dissolving polysaccharidepowder at 5 mg powder/mL solution in deuterium oxide (D₂O) containing0.01% dimethyl sulfoxide (DMSO) and 0.01%2,2-Dimethyl-2-silapentane-5-sulfonate-d₆ sodium salt (DSS-d₆). DMSO isan internal standard that was used for quantitative analysis and DSS-d₆was used to set the chemical shift scale to 0 ppm. A one-dimensionalproton NMR data set was acquired at 50° C. and a portion of spectrumcontaining the anomeric resonances as then selectively written as x, ycoordinates to an ASCII file for analysis using a Microsoft Excelworkbook. The Y coordinates (i.e., spectral profile) were then comparedto spectral profiles of capsular bacterial polysaccharides in areference database. The reference profiles were generated in a similarmanner on selected preparations of each serotype thereafter designatedas the reference lot. A pairwise comparison was made of the y-valuesfrom the sample and the reference spectra to produce a correlationcoefficient (ρ_(x,y)) as a measure of similarity between the spectra. Aρ-value of ≥0.95 with any of the reference spectra was taken as positiveidentification of the polysaccharide structure.

FIGS. 1-4 provide the 600 MHz one-dimensional ¹H NMR spectra of capsularpolysaccharides from S. pneumonia serotypes 6C, 15A, de-O-acetylated 15Band 35B, respectively. The ¹H NMR identity regions used for serotypeidentification of S. pneumonia serotypes 6C, 15A, de-O-acetylated 15Band 35B are provided in FIGS. 5-8 , respectively.

Structure of Capsular Polysaccharides from S. pneumonia SerotypesDe-O-Acetylated 15B and 15C

Immunogenicity studies were conducted using PCV21 in New Zealand Whiterabbits (see EXAMPLE 43, infra). In these studies, de-O-acetylatedpolysaccharide serotype 15B was used in the polyvalent composition inplace of serotype 15C. NMR studies were conducted to confirm thatde-O-acetylated 15B polysaccharide was equivalent to serotype 15Cpolysaccharide.

Structural differences between capsular polysaccharide serotypes show upas chemical shift differences in the NMR spectrum. The anomeric region(approximately 4.4 ppm to 6.0 ppm) is sensitive to every structuralfeature in the repeat unit of the polysaccharide. Differences instereochemistry, monosaccharide composition, 0-acetylation and glyosidiclinkage impacts the chemical shifts of the anomeric signals leaving thisregion of the spectrum unique to each serotype. For de-O-acetylatedserotype 15B polysaccharide, the complete ¹H NMR spectrum is identicalto the ¹H NMR spectrum of serotype 15C polysaccharide, indicating therepeat unit of both polysaccharides consists of the same monosaccharidesubstituents and glyosidic linkage sites (see FIGS. 9B-9C and 10B-10C).It is also clearly shown that de-O-Acetylation step removed theO-Acetate group present in the 15B polysaccharide, and that there areessentially no observed O-Acetate groups left (see FIGS. 10A-10F).

Example 4

Preparation of Serotype 3 Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in dimethylsulfoxide (DMSO). Redissolved polysaccharide andCRM197 solutions were then combined and conjugated as described below.The resulting conjugate was purified by ultrafiltration prior to a final0.2-micron filtration. Several process parameters within each step, suchas pH, temperature, concentration, and time were controlled to yieldconjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 380 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.25 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 12 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 2 mgPs/mL with sucrose concentration of 10% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 1.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.3. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 1 hour at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1.6 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 1 Attributes of serotype 3 conjugate for polyvalent study fromDMSO conjugation Lysine Free Consumption Protein / Oxidized Conjugate(mol/ mol Free Ps / Total Ps Mn / Mw Mn / Mw Ps:Pr CRM197) Total PsProtein 186 / 253 kD 862/1468 kD 1.16 8.2 <1% <1%

Example 5

Preparation of Serotype 6C for Conjugate Monovalent Study Using DMSOConjugation

Polysaccharide was dissolved, chemically activated and buffer-exchangedby ultrafiltration. Activated polysaccharide and purified CRM197 wereindividually lyophilized and redissolved in DMSO. Redissolvedpolysaccharide and CRM197 solutions were then combined and conjugated asdescribed below. The resulting conjugate was purified by ultrafiltrationprior to a final 0.2-micron filtration. Several process parameterswithin each step, such as pH, temperature, concentration, and time werecontrolled to yield conjugates with desired attributes.

Polysaccharide Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Filtered dissolved polysaccharide was concentratedand diafiltered against water using a 10 kDa NMWCO tangential flowultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.10 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 2.2 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.4. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 15 hours at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 2 Attributes of serotype 6C conjugate for monovalent study fromDMSO conjugation Lysine Free Consumption Protein / Oxidized Conjugate(mol/ mol Free Ps / Total Ps Mn / Mw Mn / Mw Ps:Pr CRM197) Total PsProtein 144 / 229 kD 2038 / 4182 kD 1.20 9.2 3.1% 5.8%

Example 6

Preparation of Serotype 6C Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, chemically activated and buffer-exchangedby ultrafiltration. Activated polysaccharide and purified CRM197 wereindividually lyophilized and redissolved in DMSO. Redissolvedpolysaccharide and CRM197 solutions were then combined and conjugated asdescribed below. The resulting conjugate was purified by ultrafiltrationprior to a final 0.2-micron filtration. Several process parameterswithin each step, such as pH, temperature, concentration, and time werecontrolled to yield conjugates with desired attributes.

Polysaccharide Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Filtered dissolved polysaccharide was concentratedand diafiltered against water using a 10 kDa NMWCO tangential flowultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.10 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 1.9 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.4. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 15 hours at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Project Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 3 Attributes of serotype 6C conjugate for polyvalent study fromDMSO conjugation Lysine Free Consumption Protein / Oxidized Conjugate(mol/ mol Free Ps / Total Ps Mn / Mw Mn / Mw Ps:Pr CRM197) Total PsProtein 144 / 229 kD 2104 / 5006 kD 1.11 8.4 4.9% 2.2%Preparation of Serotype 6A Conjugate for Monovalent Studies Using DMSOConjugation

Polysaccharide was dissolved, chemically activated and buffer-exchangedby ultrafiltration. Activated polysaccharide and purified CRM197 wereindividually lyophilized and redissolved in DMSO. Redissolvedpolysaccharide and CRM197 solutions were then combined and conjugated asdescribed below. The resulting conjugate was purified by ultrafiltrationprior to a final 0.2-micron filtration. Several process parameterswithin each step, such as pH, temperature, concentration, and time werecontrolled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 200 bar/5 passes.Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.10 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 1.5 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.4. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 15 hour at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 3 Attributes of serotype 6A conjugate for monovalent study fromDMSO conjugation Lysine Free Consumption Protein/ Oxidized Conjugate(mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:Pr CRM197) Total Ps Protein238/ 2565/ 1.021 9.2 3% 5% 278 kD 6310 kD

Example 7

Preparation of Serotype 7F Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 150 bar/7 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.24 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 4 hours at 4° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 2.6 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 4 hours at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 4 Attributes of serotype 7F conjugate for polyvalent study fromDMSO conjugation Lysine Free Consumption Protein/ Oxidized Conjugate(mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:Pr CRM197) Total Ps Protein76/118 kD 1817/ 1.55 6.7 <1% 3.2% 4026 kD

Example 8

Preparation of Serotype 8 Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 600 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.18 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 4 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 4.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. After the blend, the conjugation reaction proceeded for 2hours at 22° C. Sodium cyanoborohydride was not added to the conjugationreaction because it was observed that sodium cyanoborohydride additioncould result in irreversible precipitation during the conjugationreaction for serotype 8. Omitting sodium cyanoborohydride from thereaction avoided precipitation without significantly impacting conjugateattributes.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidine150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at 4°C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

TABLE 5 Attributes of serotype 8 conjugate for polyvalent study fromDMSO conjugation Lysine Free Consumption Protein/ Oxidized Conjugate(mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:Pr CRM197) Total Ps Protein110/ 1479/ 1.17 10.3 3.7% <1% 128 kD 2196 kD

Example 9

Preparation of Serotype 9N Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 250 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.16 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 4 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.25 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 1 hour at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidine150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at 4°C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 6 Attributes of serotype 9N conjugate for polyvalent study fromDMSO conjugation Lysine Free Consumption Protein/ Oxidized Conjugate(mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:Pr CRM197) Total Ps Protein195/ 1407/ 1.33 10.1 1.9% <1% 226 kD 3134 kD

Example 10

Preparation of Serotype 10A Conjugate for Monovalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 200 bar/5 passesfollowed by 600 bar/5 passes to achieve a target molecular mass.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.15 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 5.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 2.0. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 4 hours at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidine150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at 4°C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

TABLE 7 Attributes of serotype 10A conjugate for monovalent study fromDMSO conjugation Lysine Free Consumption Protein/ Oxidized Conjugate(mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:Pr CRM197) Total Ps Protein76/ 5137/ 1.13 5.1 <1% 15% 111 kD 7061 kD

Example 11

Preparation of Serotype 10A Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 600 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.16 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 4.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.75. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 4 hours at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidine150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at 4°C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

TABLE 8 Attributes of serotype 10A conjugate for polyvalent study fromDMSO conjugation Lysine Free Consumption Protein/ Oxidized Conjugate(mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:Pr CRM197) Total Ps Protein94/ 1540/ 1.54 7.7 8.5% <1% 130 kD 3000 kD

Example 12

Preparation of Serotype 11A Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was size-reduced by acidhydrolysis by adding acetic acid to 200 mM, incubating at 92° C. for 75minutes, then neutralizing by adding cold potassium phosphate pH 7buffer to 400 mM.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.13 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO containing 25 mM sodium chloride. Thepolysaccharide and CRM197 solutions were blended to achieve apolysaccharide concentration of 3.5 g Ps/L and a polysaccharide toCRM197 mass ratio of 1.5. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Sodiumcyanoborohydride (1 mole per mole of polysaccharide repeating unit) wasadded, and conjugation proceeded for 4 hours at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

TABLE 9 Attributes of serotype 11A conjugate for polyvalent study fromDMSO conjugation Lysine Free Consumption Protein/ Oxidized Conjugate(mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:Pr CRM197) Total Ps Protein1584/ 0.89 7.4 2.2% 2.8% 63/96 kD 2804 kD

Example 13

Preparation of Serotype 12F Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was size-reduced by acidhydrolysis by adding acetic acid to 200 mM, incubating at 90° C. for 45minutes, then neutralizing by adding cold potassium phosphate pH 7buffer to 400 mM.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.26 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO containing 25 mM sodium chloride. Thepolysaccharide and CRM197 solutions were blended to achieve apolysaccharide concentration of 3.0 g Ps/L and a polysaccharide toCRM197 mass ratio of 1.5. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Sodiumcyanoborohydride (1 mole per mole of polysaccharide repeating unit) wasadded, and conjugation proceeded for 4 hours at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidine150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at 4°C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 10 Attributes of serotype 12F conjugate for polyvalent study fromDMSO conjugation Lysine Free Consumption Protein/ Oxidized Conjugate(mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:Pr CRM197) Total Ps Protein1766/ 1.20 9.6 1.3% 1.5% 54/73 kD 3119 kD

Example 14

Preparation of Serotype 15A Conjugate for Monovalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 200 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 1.75 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 20 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO containing 25 mM sodium chloride. Thepolysaccharide and CRM197 solutions were blended to achieve apolysaccharide concentration of 6.0 g Ps/L and a polysaccharide toCRM197 mass ratio of 2.0. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Sodiumcyanoborohydride (1 mole per mole of polysaccharide repeating unit) wasadded, and conjugation proceeded for 2.5 hours at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidine150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at 4°C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

TABLE 11 Attributes of serotype 15A conjugate for monovalent study fromDMSO conjugation Lysine Free Consumption Protein/ Oxidized Conjugate(mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:Pr CRM197) Total Ps Protein158/ 6949/ 1.05 9.7 10% 5.0% 200 kD 9235 kD

Example 15

Preparation of Serotype 15A Conjugate for 15A/B/C Cross Protection StudyUsing Aqueous Conjugation

Polysaccharide was dissolved, size reduced, chemically activated andbuffer-exchanged by ultrafiltration. Purified CRM197 was then conjugatedto the activated polysaccharide utilizing nickel chloride in the aqueousreaction mixture, and the resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular polysaccharide powder was dissolved inwater and 0.45-micron filtered. Dissolved polysaccharide was homogenizedto reduce the molecular mass. Homogenization pressure and number ofpasses through the homogenizer were controlled to 200 bar/5 passes toachieve a target molecular mass. Size-reduced polysaccharide was thenconcentrated and diafiltered against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.45 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit. The oxidation reaction proceeded for 20hours at 22° C. The activated product was diafiltered against 10 mMpotassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flowultrafiltration membrane. Ultrafiltration was conducted at 2-8° C.Further activation was achieved by adjusting the ultrafiltered productto 22° C. and pH 5. Activation was conducted with the addition of 100 mMsodium metaperiodate solution with a charge of 2.0 moles of sodiummetaperiodate per mole of polysaccharide repeating unit to achieve atarget level of polysaccharide activation (moles aldehyde per mole ofpolysaccharide repeating unit). The oxidation reaction proceeded for 20hours at 22° C. The second activated product was diafiltered against 10mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flowultrafiltration membrane. Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Oxidized polysaccharide solution was mixed with water and 1.5 Mpotassium phosphate pH 6.0. The buffer pH selected was to improve thestability of activated polysaccharide during the conjugation reaction.Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was 0.2-micron filtered andcombined with the buffered polysaccharide solution at a polysaccharideto CRM197 mass ratio of 0.6. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Thepolysaccharide and phosphate concentrations were 9.75 g/L and 100 mMrespectively. The polysaccharide concentration was selected to controlthe size of the resulting conjugate. Nickel chloride was added toapproximately 2 mM using a 100 mM nickel chloride solution. Sodiumcyanoborohydride (2 moles per mole of polysaccharide repeating unit) wasadded. Conjugation proceeded for 148 hours at 10° C. to maximizeconsumption of polysaccharide and protein.

Reduction with Sodium Borohydride

Following the conjugation reaction, the batch was diluted to apolysaccharide concentration of approximately 3.0 g/L, cooled to 2-8°C., and 1.2-micron filtered. The batch was diafiltered against 100 mMpotassium phosphate, pH 7.0 at 2-8° C. using a 100 kDa NMWCO tangentialflow ultrafiltration membrane. The batch, recovered in the retentate,was then diluted to approximately 2.0 g polysaccharide/L and pH-adjustedwith the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodiumborohydride (1 mole per mole of polysaccharide repeating unit) wasadded. 1.5 M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mML-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDaNMWCO tangential flow ultrafiltration membrane. Polysorbate 20 was addedto the retentate batch to a concentration of 0.05% (w/v) then the batchwas 0.2 micron filtered (with 0.5 micron prefilter).

The batch was adjusted to a polysaccharide concentration of 1.0 g/L withadditional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 bufferwith 0.03% (w/v) polysorbate 20. The batch was dispensed into aliquotsand frozen at ≤−60° C.

TABLE 12 Attributes of serotype 15A conjugate for 15A/B/C crossprotection study from aqueous conjugation Lysine Free ConsumptionProtein/ Oxidized Conjugate (mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:PrCRM197) Total Ps Protein 118/ 465/ 0.99 4.5 9.6% <1% 154 kD 695 kD

Example 16

Preparation of Serotype 15A Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 200 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 1.75 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 20 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO which was pre-heated to 34° C. The polysaccharidesolution was spiked with sodium chloride to a concentration of 50 mM.The polysaccharide and CRM197 solutions were blended to achieve apolysaccharide concentration of 5.0 g Ps/L and a polysaccharide toCRM197 mass ratio of 2.0. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Sodiumcyanoborohydride (1 mole per mole of polysaccharide repeating unit) wasadded, and conjugation proceeded for 2 hours at 34° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 13 Attributes of serotype 15A conjugate for polyvalent study fromDMSO conjugation Lysine Free Consumption Protein/ Oxidized Conjugate(mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:Pr CRM197) Total Ps Protein214/ 1628/ 1.57 7.3 17% 7.8% 231 kD 3518 kD

Example 17

Preparation of Serotype 15B Conjugate for 15A/B/C Cross Protection StudyUsing DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 300 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.20 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 4 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 2.0. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 5 hours at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

TABLE 14 Attributes of serotype 15B conjugate for 15A/B/C crossprotection study from DMSO conjugation Lysine Free Consumption Protein/Oxidized Conjugate (mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:Pr CRM197)Total Ps Protein 203/ 2329/ 1.67 7.9 3.6% 4.4% 252 kD 3881 kD

Example 18

Preparation of Serotype 15C Conjugate for Monovalent Study and 15A/B/CCross Protection Study Using DMSO Conjugation

Polysaccharide derived from Streptococcus pneumoniae serotype 15B wasdissolved, sized to a target molecular mass, subjected to mild basehydrolysis to release O-acetyl groups, chemically activated andbuffer-exchanged by ultrafiltration. Activated polysaccharide andpurified CRM197 were individually lyophilized and redissolved in DMSO.Redissolved polysaccharide and CRM197 solutions were then combined andconjugated as described below. The resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction, Base Hydrolysis and Oxidation

Purified serotype 15B pneumococcal capsular Ps powder was dissolved inwater and 0.45-micron filtered. Dissolved polysaccharide was homogenizedto reduce the molecular mass of the Ps. Homogenization pressure andnumber of passes through the homogenizer were controlled to 300 bar/5passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was heated to 60° C. and sodium bicarbonatepH 9 buffer was added to a final concentration 50 mM. The batch wasincubated with mixing for 13 hours at 60° C. to release O-acetyl groups.Potassium phosphate pH 6 buffer was added to a final concentration of136 mM to neutralize pH and the solution was cooled to ambienttemperature. The solution was then concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.20 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 2.0. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 4 hours at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

TABLE 15 Attributes of serotype 15C conjugate for monovalent study and15A/B/C cross protection study from DMSO conjugation Oxidized ConjugateLysine Consumption Free Ps/ Free Protein/ O-acetyl/Ps Ps Mn/Mw Mn/MwPs:Pr (mol/mol CRM197) Total Ps Total Protein None 182/246 1962/40191.31 4.8 3.8% 13% detected kD kD

Example 19

Preparation of Serotype 15C Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide derived from Streptococcus pneumoniae serotype 15B wasdissolved, sized to a target molecular mass, subjected to mild basehydrolysis to release O-acetyl groups, chemically activated andbuffer-exchanged by ultrafiltration. Activated polysaccharide andpurified CRM197 were individually lyophilized and redissolved in DMSO.Redissolved polysaccharide and CRM197 solutions were then combined andconjugated as described below. The resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction, Base Hydrolysis and Oxidation

Purified serotype 15B pneumococcal capsular Ps powder was dissolved inwater and 0.45-micron filtered. Dissolved polysaccharide was homogenizedto reduce the molecular mass of the Ps. Homogenization pressure andnumber of passes through the homogenizer were controlled to 300 bar/5passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was heated to 60° C. and sodium bicarbonatepH 9 buffer was added to a final concentration 50 mM. The batch wasincubated with mixing for 13 hours at 60° C. to release O-acetyl groups.Potassium phosphate pH 6 buffer was added to a final concentration of136 mM to neutralize pH and the solution was cooled to ambienttemperature. The solution was then concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.20 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.75. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 8 hours at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

TABLE 16 Attributes of serotype 15C conjugate ffor polyvalent study romDMSO conjugation Oxidized Conjugate Lysine Consumption Free Ps/ FreeProtein/ O-acetyl/Ps Ps Mn/Mw Mn/Mw Ps:Pr (mol/mol CRM197) Total PsTotal Protein None 182/246 1540/3230 1.34 6.6 9.2% 5.7% detected kD kD

Example 20

Preparation of Serotype 16F Conjugate for Monovalent Study Using AqueousConjugation

Polysaccharide was dissolved, size reduced, chemically activated andbuffer-exchanged by ultrafiltration. Purified CRM197 was then conjugatedto the activated polysaccharide utilizing nickel chloride in the aqueousreaction mixture, and the resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular polysaccharide powder was dissolved inwater, and 0.45-micron filtered. Dissolved polysaccharide washomogenized to reduce the molecular mass. Homogenization pressure andnumber of passes through the homogenizer were controlled to 200 bar/5passes followed by 500 bar/5 passes to achieve a target molecular mass.Size-reduced polysaccharide was then concentrated and diafilteredagainst water using a 10 kDa NMWCO tangential flow ultrafiltrationmembrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.15 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Oxidized polysaccharide solution was mixed with water and 1.5 Mpotassium phosphate pH 7.0. The buffer pH selected was to improve thestability of activated polysaccharide during the conjugation reaction.Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was 0.2-micron filtered andcombined with the buffered polysaccharide solution at a polysaccharideto CRM197 mass ratio of 0.7. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Thepolysaccharide and phosphate concentrations were 7.5 g/L and 100 mMrespectively. The polysaccharide concentration was selected to controlthe size of the resulting conjugate. The solution was then 0.2-micronfiltered. Nickel chloride was added to approximately 2 mM using a 100 mMnickel chloride solution. Sodium cyanoborohydride (2 moles per mole ofpolysaccharide repeating unit) was added. Conjugation proceeded for 122hours at 22° C. to maximize consumption of polysaccharide and protein.

Reduction with Sodium Borohydride

Following the conjugation reaction, the batch was diluted to apolysaccharide concentration of approximately 3.0 g/L, cooled to 2-8°C., and 1.2-micron filtered. The batch was diafiltered against 100 mMpotassium phosphate, pH 7.0 at 2-8° C. using a 100 kDa NMWCO tangentialflow ultrafiltration membrane. The batch, recovered in the retentate,was then diluted to approximately 2.0 g polysaccharide/L and pH-adjustedwith the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodiumborohydride (1 mole per mole of polysaccharide repeating unit) wasadded. 1.5 M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mML-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDaNMWCO tangential flow ultrafiltration membrane. Polysorbate 20 was addedto the retentate batch to a concentration of 0.05% (w/v) then the batchwas 0.2 micron filtered (with 0.5 micron prefilter).

The batch was adjusted to a polysaccharide concentration of 1.0 g/L withadditional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 bufferwith 0.03% (w/v) polysorbate 20. The batch was dispensed into aliquotsand frozen at ≤−60° C.

TABLE 17 Attributes of serotype 16F conjugate for monovalent study fromaqueous conjugation Lysine Free Consumption Protein/ Oxidized Conjugate(mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:Pr CRM197) Total Ps Protein90/ 1860/ 1.10 3.3 7.0% <1% 139 kD 5539 kD

Example 21

Preparation of Serotype 16F Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 1000 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.15 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a concentration of 50 mM. The polysaccharide andCRM197 solutions were blended to achieve a polysaccharide concentrationof 2.0 g Ps/L and a polysaccharide to CRM197 mass ratio of 1.5. The massratio was selected to control the polysaccharide to CRM197 ratio in theresulting conjugate. Sodium cyanoborohydride (1 mole per mole ofpolysaccharide repeating unit) was added, and conjugation proceeded for2 hours at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 18 Attributes of serotype 16F conjugate for polyvalent study fromDMSO conjugation Lysine Free Consumption Protein/ Oxidized Conjugate(mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:Pr CRM197) Total Ps Protein91/ 2075/ 1.29 11.0 <1% <1% 177 kD 3966 kD

Example 22

Preparation of Serotype 17F Conjugate for Monovalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 200 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.11 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a concentration of 50 mM. The polysaccharide andCRM197 solutions were blended to achieve a polysaccharide concentrationof 2.0 g Ps/L and a polysaccharide to CRM197 mass ratio of 1.5. The massratio was selected to control the polysaccharide to CRM197 ratio in theresulting conjugate. Sodium cyanoborohydride (1 mole per mole ofpolysaccharide repeating unit) was added, and conjugation proceeded for2 hours at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 19 Attributes of serotype 17F conjugate for monovalent study fromDMSO conjugation Lysine Free Consumption Protein/ Oxidized Conjugate(mol/mol Free Ps/ Total Ps Mn/Mw Mn/Mw Ps:Pr CRM197) Total Ps Protein179/ 2630/ 1.20 8.0 1.9% 4.5% 216 kD 4632 kD

Example 23

Preparation of Serotype 17F Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 200 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.11 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a concentration of 20 mM. The polysaccharide andCRM197 solutions were blended to achieve a polysaccharide concentrationof 2.1 g Ps/L and a polysaccharide to CRM197 mass ratio of 1.5. The massratio was selected to control the polysaccharide to CRM197 ratio in theresulting conjugate. Sodium cyanoborohydride (1 mole per mole ofpolysaccharide repeating unit) was added, and conjugation proceeded for2 hours at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidine150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at 4°C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

TABLE 20 Attributes of serotype 17F conjugate for polyvalent study fromDMSO conjugation Lysine Free Oxidized Consumption Protein/ Ps Mn/Conjugate (mol/mol Free Ps/ Total Mw Mn/Mw Ps:Pr CRM197) Total PsProtein 150/212 2650/ 1.10 7.7 2.8% 1.5% kD 4110 kD

Example 24

Preparation of Serotype 19A Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, chemically activated and buffer-exchangedby ultrafiltration. Activated polysaccharide and purified CRM197 wereindividually lyophilized and redissolved in DMSO. Redissolvedpolysaccharide and CRM197 solutions were then combined and conjugated asdescribed below. The resulting conjugate was purified by ultrafiltrationprior to a final 0.2-micron filtration. Several process parameterswithin each step, such as pH, temperature, concentration, and time werecontrolled to yield conjugates with desired attributes.

Polysaccharide Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. The polysaccharide was concentrated anddiafiltered against water using a 10 kDa NMWCO tangential flowultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The amount of sodiummetaperiodate added was 0.26 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The oxidation reaction proceeded for 20 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.8 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.33. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 1.5 hours at22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 21 Attributes of serotype 19A conjugate for polyvalent study fromDMSO conjugation Lysine Free Oxidized Consumption Protein/ Ps Mn/Conjugate (mol/mol Free Ps/ Total Mw Mn/Mw Ps:Pr CRM197) Total PsProtein 96/186 1714/ 1.22 9.1 7.0% 1.4% kD 3585 kD

Example 25

Preparation of Serotype 20A Conjugate for Monovalent Study Using DMSOConjugation Polysaccharide previously determined to be serotype 20A(Calix et al., Biochemical, Genetic, and Serological Characterization ofTwo Capsule Subtypes among Streptococcus pneumoniae Serotype 20 Strains,J Biol. Chem. 287(33): 27885-27894, (2012)) was dissolved, sized to atarget molecular mass, chemically activated and buffer-exchanged byultrafiltration. Activated polysaccharide and purified CRM197 wereindividually lyophilized and redissolved in DMSO. Redissolvedpolysaccharide and CRM197 solutions were then combined and conjugated asdescribed below. The resulting conjugate was purified by ultrafiltrationprior to a final 0.2-micron filtration. Several process parameterswithin each step, such as pH, temperature, concentration, and time werecontrolled to yield conjugates with desired attributes.Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 200 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. Sodium metaperiodate added at0.11 moles of sodium metaperiodate per mole of polysaccharide repeatingunit to achieve a target level of polysaccharide activation (molesaldehyde per mole of polysaccharide repeating unit). The oxidationreaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 2.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 6 hours at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidine150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at 4°C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

TABLE 22 Attributes of serotype 20A conjugate for monovalent study fromDMSO conjugation Lysine Free Oxidized Consumption Protein/ Ps Mn/Conjugate (mol/mol Free Ps/ Total Mw Mn/Mw Ps:Pr CRM197) Total PsProtein 165/215 5159/ 1.17 6.1 <1% 6.5% kD 7778 kD

Example 26

Preparation of Serotype 20A Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 220 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. Sodium metaperiodate added at0.16 moles of sodium metaperiodate per mole of polysaccharide repeatingunit to achieve a target level of polysaccharide activation (molesaldehyde per mole of polysaccharide repeating unit). The oxidationreaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a concentration of 20 mM. The polysaccharide andCRM197 solutions were blended to achieve a polysaccharide concentrationof 1.4 g Ps/L and a polysaccharide to CRM197 mass ratio of 1.5. The massratio was selected to control the polysaccharide to CRM197 ratio in theresulting conjugate. Sodium cyanoborohydride (1 mole per mole ofpolysaccharide repeating unit) was added, and conjugation proceeded for4 hours at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidine150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at 4°C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

TABLE 23 Attributes of serotype 20A conjugate for polyvalent study fromDMSO conjugation Lysine Free Oxidized Consumption Protein/ Ps Mn/Conjugate (mol/mol Free Ps/ Total Mw Mn/Mw Ps:Pr CRM197) Total PsProtein 178/215 2478/ 1.10 8.3 1.0% 1.0% kD 3863 kD

Example 27

Preparation of Serotype 22F Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 400 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. Sodium metaperiodate added at0.12 moles of sodium metaperiodate per mole of polysaccharide repeatingunit to achieve a target level of polysaccharide activation (molesaldehyde per mole of polysaccharide repeating unit). The oxidationreaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 1.8 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 1 hour at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidine150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at 4°C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 24 Attributes of serotype 22F conjugate for polyvalent study fromDMSO conjugation Lysine Free Oxidized Consumption Protein/ Ps Mn/Conjugate (mol/mol Free Ps/ Total Mw Mn/Mw Ps:Pr CRM197) Total PsProtein 170/196 1800/ 1.13 7.0 <1% 1.1% kD 3970 kD

Example 28

Preparation of Serotype 23A Conjugate for Monovalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was size-reduced by acidhydrolysis by adding acetic acid to 200 mM, incubating at 90° C. for 1.5hours, then neutralizing by adding cold potassium phosphate pH 7 bufferto 400 mM.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The sodium metaperiodate addedwas 0.20 moles of sodium metaperiodate per mole of polysacchariderepeating unit to achieve a target level of polysaccharide activation(moles aldehyde per mole of polysaccharide repeating unit). Theoxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a concentration of 50 mM. The polysaccharide andCRM197 solutions were blended to achieve a polysaccharide concentrationof 3.0 g Ps/L and a polysaccharide to CRM197 mass ratio of 1.5. The massratio was selected to control the polysaccharide to CRM197 ratio in theresulting conjugate. Sodium cyanoborohydride (1 mole per mole ofpolysaccharide repeating unit) was added and conjugation proceeded for 2hours at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 25 Attributes of serotype 23A conjugate for monovalent study fromDMSO conjugation Lysine Free Oxidized Consumption Protein/ Ps Mn/Conjugate (mol/mol Free Ps/ Total Mw Mn/Mw Ps:Pr CRM197) Total PsProtein 70/97 2183/ 1.20 11.8 <1% <1% kD 3837 kD

Example 29

Preparation of Serotype 23A Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was size-reduced by acidhydrolysis by adding acetic acid to 200 mM, incubating at 90° C. for 1.5hours, then neutralizing by adding cold potassium phosphate pH 7 bufferto 400 mM.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The sodium metaperiodate addedwas 0.20 moles of sodium metaperiodate per mole of polysacchariderepeating unit to achieve a target level of polysaccharide activation(moles aldehyde per mole of polysaccharide repeating unit). Theoxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.5 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added and conjugation proceeded for 2 hours at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 26 Attributes of serotype 23A conjugate for polyvalent study fromDMSO conjugation Lysine Free Oxidized Consumption Protein/ Ps Mn/Conjugate (mol/mol Free Ps/ Total Mw Mn/Mw Ps:Pr CRM197) Total PsProtein 116/175 2156/ 1.12 6.3 3.3% 2.1% kD 4933 kD

Example 30

Preparation of Serotype 23B Conjugate for Monovalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 400 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. Sodium metaperiodate added at0.10 moles of sodium metaperiodate per mole of polysaccharide repeatingunit to achieve a target level of polysaccharide activation (molesaldehyde per mole of polysaccharide repeating unit). The oxidationreaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a final concentration of 50 mM. The polysaccharideand CRM197 solutions were blended to achieve a polysaccharideconcentration of 5.0 g Ps/L and a polysaccharide to CRM197 mass ratio of1.5. The mass ratio was selected to control the polysaccharide to CRM197ratio in the resulting conjugate. Sodium cyanoborohydride (1 mole permole of polysaccharide repeating unit) was added, and conjugationproceeded for 2 hours at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 27 Attributes of serotype 23B conjugate for monovalent study fromDMSO conjugation Lysine Free Oxidized Consumption Protein/ Ps Mn/Conjugate (mol/mol Free Ps/ Total Mw Mn/Mw Ps:Pr CRM197) Total PsProtein 155/179 1322/ 1.28 6.2 13% 5.1% kD 3299 kD

Example 31

Preparation of Serotype 23B Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 400 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. Sodium metaperiodate added at0.13 moles of sodium metaperiodate per mole of polysaccharide repeatingunit to achieve a target level of polysaccharide activation (molesaldehyde per mole of polysaccharide repeating unit). The oxidationreaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 5.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 4 hours at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 28 Attributes of serotype 23B conjugate for polyvalent study fromDMSO conjugation Lysine Free Oxidized Consumption Protein/ Ps Mn/Conjugate (mol/mol Free Ps/ Total Mw Mn/Mw Ps:Pr CRM197) Total PsProtein 172/197 1076/ 1.26 7.4 12% 3.2% kD 2514 kD

Example 32

Preparation of Serotype 24F Conjugate for Monovalent and PolyvalentStudies Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was size-reduced by acidhydrolysis by adding acetic acid to 200 mM, incubating at 92° C. for 50minutes, then neutralizing by adding cold potassium phosphate pH 7buffer to 400 mM.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The sodium metaperiodate addedwas 0.18 moles of sodium metaperiodate per mole of polysacchariderepeating unit to achieve a target level of polysaccharide activation(moles aldehyde per mole of polysaccharide repeating unit). Theoxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 2 mgPs/mL with sucrose concentration of 10% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a final concentration of 50 mM. The polysaccharideand CRM197 solutions were blended to achieve a polysaccharideconcentration of 1.5 g Ps/L and a polysaccharide to CRM197 mass ratio of1.5. The mass ratio was selected to control the polysaccharide to CRM197ratio in the resulting conjugate. Sodium cyanoborohydride (1 mole permole of polysaccharide repeating unit) was added, and conjugationproceeded for 2 hours at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 29 Attributes of serotype 24F conjugate for monovalent andpolyvalent studies from DMSO conjugation Lysine Free OxidizedConsumption Protein/ Ps Mn/ Conjugate (mol/mol Free Ps/ Total Mw Mn/MwPs:Pr CRM197) Total Ps Protein 56/100 2233/ 0.94 7.3 4.4% 1.4% kD 4875kD

Example 33

Preparation of Serotype 31 Conjugate for Monovalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was size-reduced by acidhydrolysis by adding acetic acid to 200 mM, incubating at 90° C. for 30minutes, then neutralizing by adding cold potassium phosphate pH 7buffer to 400 mM.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The sodium metaperiodate addedwas 0.16 moles of sodium metaperiodate per mole of polysacchariderepeating unit to achieve a target level of polysaccharide activation(moles aldehyde per mole of polysaccharide repeating unit). Theoxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 4.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added and conjugation proceeded for 1 hour at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 30 Attributes of serotype 31 conjugate for monovalent study fromDMSO conjugation Lysine Free Oxidized Consumption Protein/ Ps Mn/Conjugate (mol/mol Free Ps/ Total Mw Mn/Mw Ps:Pr CRM197) Total PsProtein 96/119 1818/ 1.15 9.5 <1% <1% kD 2999 kD

Example 34

Preparation of Serotype 31 Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 400 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. Sodium metaperiodate added at0.12 moles of sodium metaperiodate per mole of polysaccharide repeatingunit to achieve a target level of polysaccharide activation (molesaldehyde per mole of polysaccharide repeating unit). The oxidationreaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a final concentration of 50 mM. The polysaccharideand CRM197 solutions were blended to achieve a polysaccharideconcentration of 3.5 g Ps/L and a polysaccharide to CRM197 mass ratio of1.5. The mass ratio was selected to control the polysaccharide to CRM197ratio in the resulting conjugate. Sodium cyanoborohydride (1 mole permole of polysaccharide repeating unit) was added, and conjugationproceeded for 1 hour at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

TABLE 31 Attributes of serotype 31 conjugate for polyvalent study fromDMSO conjugation Lysine Free Oxidized Consumption Protein/ Ps Mn/Conjugate (mol/mol Free Ps/ Total Mw Mn/Mw Ps:Pr CRM197) Total PsProtein 149/186 2354/ 1.26 8.9 <1% <1.8% kD 5971 kD

Example 35

Preparation of Serotype 33F Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 350 bar/4 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. Sodium metaperiodate was addedat 0.12 moles of sodium metaperiodate per mole of polysacchariderepeating unit to achieve a target level of polysaccharide activation(moles aldehyde per mole of polysaccharide repeating unit). Theoxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 1.8 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.75. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded for 4 hours at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

TABLE 32 Attributes of serotype 33F conjugate for polyvalent study fromDMSO conjugation Lysine Free Oxidized Consumption Protein/ Ps Mn/Conjugate (mol/mol Free Ps/ Total Mw Mn/Mw Ps:Pr CRM197) Total PsProtein 186/220 1630/ 1.28 8.4 1.3% 5.4% kD 2470 kD

Example 36

Preparation of Serotype 35B Conjugate for Monovalent Study Using DMSOConjugation

Polysaccharide was dissolved, chemically activated and buffer-exchangedby ultrafiltration. Activated polysaccharide and purified CRM197 wereindividually lyophilized and redissolved in DMSO. Redissolvedpolysaccharide and CRM197 solutions were then combined and conjugated asdescribed below. The resulting conjugate was purified by ultrafiltrationprior to a final 0.2-micron filtration. Several process parameterswithin each step, such as pH, temperature, concentration, and time werecontrolled to yield conjugates with desired attributes.

Polysaccharide Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was concentrated anddiafiltered against water using a 10 kDa NMWCO tangential flowultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The sodium metaperiodate addedwas 0.05 moles of sodium metaperiodate per mole of polysacchariderepeating unit to achieve a target level of polysaccharide activation(moles aldehyde per mole of polysaccharide repeating unit). Theoxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 were redissolved individually in equal volumesof DMSO containing 25 mM sodium chloride. The polysaccharide and CRM197solutions were blended to achieve a polysaccharide concentration of 10 gPs/L and a polysaccharide to CRM197 mass ratio of 4.0. The mass ratiowas selected to control the polysaccharide to CRM197 ratio in theresulting conjugate. Conjugation proceeded for 2.5 hours at 22° C.Sodium borohydride (0.025 moles per mole of polysaccharide repeatingunit) was added over two spikes during the conjugation incubation.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1.5 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

TABLE 33 Attributes of serotype 35B conjugate for monovalent study fromDMSO conjugation Lysine Free Oxidized Consumption Protein/ Ps Mn/Conjugate (mol/mol Free Ps/ Total Mw Mn/Mw Ps:Pr CRM197) Total PsProtein 48/82 5494/ 1.26 4.5 1.8% 11% kD 7385 kD

Example 37

Preparation of Serotype 35B Conjugate for Polyvalent Study Using DMSOConjugation

Polysaccharide was dissolved, chemically activated and buffer-exchangedby ultrafiltration. Activated polysaccharide and purified CRM197 wereindividually lyophilized and redissolved in DMSO. Redissolvedpolysaccharide and CRM197 solutions were then combined and conjugated asdescribed below. The resulting conjugate was purified by ultrafiltrationprior to a final 0.2-micron filtration. Several process parameterswithin each step, such as pH, temperature, concentration, and time werecontrolled to yield conjugates with desired attributes.

Polysaccharide Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was concentrated anddiafiltered against water using a 10 kDa NMWCO tangential flowultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The sodium metaperiodate addedwas 0.05 moles of sodium metaperiodate per mole of polysacchariderepeating unit to achieve a target level of polysaccharide activation(moles aldehyde per mole of polysaccharide repeating unit). Theoxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps was redissolved in DMSO containing 50 mM sodium chloride.Lyophilized CRM197 was redissolved in an equal volume of DMSO. Thepolysaccharide and CRM197 solutions were blended to achieve apolysaccharide concentration of 5.25 g Ps/L and a polysaccharide toCRM197 mass ratio of 3.5. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Conjugationproceeded for 3 hours at 34° C. Sodium borohydride (0.0375 moles permole of polysaccharide repeating unit) was added over three spikesduring the conjugation incubation.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at34° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

TABLE 34 Attributes of serotype 35B conjugate for polyvalent study fromDMSO conjugation Lysine Free Oxidized Consumption Protein/ Ps Mn/Conjugate (mol/mol Free Ps/ Total Mw Mn/Mw Ps:Pr CRM197) Total PsProtein 48/82 1287/ 2.1 6.9 7.8% 4.6% kD 2585 kD

Some aqueous conjugation methods were not described for all serotypes inExamples 38-51. For those aqueous conjugation methods not describedherein, similar methods can be found in WO2018/156491.

Example 38

Formulation of Pneumococcal Conjugate Vaccines

Individual pneumococcal polysaccharide-protein conjugates preparedutilizing different chemistries as described in the Examples, supra,were used for the formulation of 1-, 7-, 8-, 14-, 15-, 16-, 21- and31-valent pneumococcal conjugate vaccines referred to as PCV1, PCV7,PCV8, PCV14, PCV15, PCV16, PCV21, and PCV31, respectively.

The PCV1 vaccine drug product formulation contained serotype 3conjugated either using reductive amination in a protic (aqueous)solvent or an aprotic (DMSO) solvent and formulated in 20 mM L-HistidinepH 5.8, 150 mM NaCl and 0.1% (w/v) PS-20 for a target finalconcentration of 84 μg/mL (w/v) Pneumococcal Polysaccharide (PnPs) inthe vaccine.

The PCV7 vaccine drug product formulation contained serotypes 3, 8, 9N,10A, 11A, 15A and 19A, conjugated either using reductive amination in aprotic (aqueous) solvent or an aprotic (e.g. DMSO) and formulated in 20mM L-Histidine pH 5.8, 150 mM NaCl and 0.1% (w/v) PS-20. Eachpolysaccharide-protein conjugate in PCV7 was formulated at 12 μg/mL(w/v) Pneumococcal Polysaccharide (PnPs) for a target finalconcentration of 84 μg/mL PnPs in the vaccine.

The PCV8 vaccine drug product contained serotypes 6C, 15A, 16F, 23A,23B, 24F, 31 and 35B conjugated either using reductive amination in anaprotic solvent (e.g DMSO) and formulated in 20 mM L-Histidine pH 5.8,150 mM NaCl and 0.2% (w/v) PS-20. Each polysaccharide-protein conjugatein PCV8 was formulated at 4 μg/mL (w/v) Pneumococcal Polysaccharide(PnPs) for a target final concentration of 32 μg/mL PnPs in the vaccine.

The PCV14 vaccine drug product contained serotypes 3, 7F, 8, 9N, 10A,11A, 12F, 15A, 16F, 17F, 19A, 20A, 22F and 33F conjugated either usingreductive amination in a protic (aqueous) solvent or an aprotic (e.g.DMSO) and formulated in 20 mM L-Histidine pH 5.8, 150 mM NaCl and 0.1%(w/v) PS-20. Each polysaccharide-protein conjugate in PCV14 wasformulated at 6 μg/mL (w/v) Pneumococcal Polysaccharide (PnPs) for atarget final concentration of 84 μg/mL PnPs in the vaccine.

The PCV15 vaccine drug product contained serotypes 1, 3, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated in a protic(aqueous) solvent and formulated in 20 mM L-Histidine pH 5.8, 150 mMNaCl and 0.2% (w/v) PS-20. Each polysaccharide-protein conjugate wasformulated at 4 μg/mL (w/v) Pneumococcal Polysaccharide (PnPs), exceptfor 6B, which was formulated at 8 μg/mL, for a target finalconcentration of 64 μg/mL PnPs in the vaccine.

The PCV16 vaccine drug product contained serotypes 6C, 8, 9N, 10A, 11A,12F, 15A, 15C, 16F, 17F, 20A, 23A, 23B, 24F, 31, and 35B, conjugatedusing reductive amination in an aprotic solvent (e.g. DMSO) andformulated in 20 mM L-Histidine pH 5.8 150 mM NaCl and 0.2% (w/v) PS-20.Each polysaccharide-protein conjugate was formulated at 4 μg/mL (w/v) or8 μg/mL (w/v) Pneumococcal Polysaccharide (PnPs) for a target finalconcentration of 64 μg/mL or 128 μg/mL PnPs in the vaccine.

The PCV21 vaccine drug product contained serotypes 3, 6C, 7F, 8, 9N,10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33Fand 35B, conjugated using reductive amination in an aprotic solvent(e.g. DMSO) and formulated in 20 mM L-Histidine pH 5.8 150 mM NaCl andvarious concentration of PS-20 as defined in each example. Eachpolysaccharide-protein conjugate was formulated at 4 μg/mL (w/v) or 8μg/mL (w/v) Pneumococcal Polysaccharide (PnPs) for a target finalconcentration of 84 μg/mL or 168 μg/mL PnPs in the vaccine. In someexamples, PCV21 was formulated with 250 μg [Al]/mL in the form ofAluminum Phosphate.

The PCV31 vaccine drug product contained serotypes 1, 3, 4, 5, 6A, 6B,6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 19A, 19F,20A, 22F, 23A, 23B, 23F, 24F, 31, 33F and 35B conjugated using reductiveamination in a protic solvent (aqueous) or an aprotic solvent (e.g.DMSO) and formulated in 20 mM L-Histidine pH 5.8 150 mM NaCl and 0.2%PS-20. Each polysaccharide-protein conjugate was formulated at 4 μg/mL(w/v) Pneumococcal Polysaccharide (PnPs), except for 6B, which wasformulated at 8 μg/mL PnPs, for a target final concentration of 128μg/mL PnPs in the vaccine. In some examples, PCV31 was formulated with250 μg [Al]/mL in the form of Aluminum Phosphate.

Additional monovalent PCV drug product formulations were preparedutilizing pneumococcal polysaccharide-protein conjugate serotypes 6A,6B, 6C, 10A, 15A, 15B, 15C, 16F, 17F, 20A, 23A, 23B, 24F, 31 or 35B.Additional details are described in the specific examples for theseformulations.

To prepare the PCV drug product formulation, the required volumes ofbulk conjugates needed to obtain the indicated final concentration of(w/v) Pneumococcal Polysaccharide (also referred to as PnPs) werecalculated based on the batch volume and the bulk polysaccharideconcentration.

The formulation process consisted of a conjugate bulk blend preparationat 1× to 4× of final concentration of PnPs blends in 10-80 mM Histidine,0.0-0.8% (w/v) PS-20, and 150 mM sodium chloride, pH 5.8.

Histidine pH 5.8, PS-20 (if utilized) and 150 mM sodium chloridesolutions were prepared and added to the formulation vessel. Theindividual pneumococcal polysaccharide-protein conjugates, storedfrozen, were thawed at 2-8° C. and then added to the formulation vessel.During the addition of polysaccharide-protein conjugate to theformulation buffer (conjugate blend), the vessel was mixed to ensurehomogeneity using a magnetic sir bar or magnetic impeller. After alladditions were made and the solution was stirred, the conjugate blendwas passed through sterilizing filters and collected in a vessel with orwithout Aluminum phosphate adjuvant. In some cases, the sterilizingfilters were chased with 150 mM sodium chloride to adjust the batch totarget concentration.

The formulations were filled into plastic syringes, glass syringes, orvials.

Example 39

Stability Assessment of Pneumococcal Conjugate Vaccines

The PCV16 (128 μg/mL PnPs) or two PCV21 pneumococcal conjugate vaccinedrug products (84 μg/mL or 168 μg/mL PnPs) were filled in syringes orvials. The polysaccharide protein conjugates were made via reductiveamination in an aprotic solvent (e.g. DMSO) and were formulated in 20 mML-Histidine, pH 5.8, 150 mM NaCl, and 0.2% (w/v) PS-20. These drugproducts were placed at 25° C. or 37° C. for up to four weeks and at 4°C. for up to twelve weeks. In some cases, the syringes were placed on ahorizontal rotation platform and shaken for up to 18 hours after storagefor 1 week at 4° C., 25° C. or 37° C. This was done to simulate theshipping and handling stresses that may accompany drug productmanufacture and distribution. To assess stability of the PCV16 or PCV21drug products, HPSEC UV/MALS/RI was used. High Performance SizeExclusion Chromatography (HPSEC), using two Shodex columns (803 and806), coupled together in series and Ultraviolet (UV) multi-angle lightscattering (MALS) and refractive index (RI) detection to measureconcentration and molar mass of PCV drug product formulations duringstorage. The concentration is calculated for each time interval acrossthe peak and is then integrated at all intervals to achieve the finalconcentration value. Light scattering is proportional to the product ofthe molecular mass and the concentration of the analyte. Molar masses orMolecular weight at each interval is calculated from the detectorsignals. The weighted average (Mw) or number average (Mn) molecular massis calculated across all intervals for the peak and reported as averagemolecular weight.

Storage may result in damage to protein and/or carbohydrate of aconjugate vaccine. Carbohydrate antigens that possess phosphodiestermain-chain bonds or other instabilities in the repeat unit of thepolysaccharide may be susceptible to depolymerization through hydrolysisupon storage for extended time and temperature. Furthermore, the carrierprotein or carbohydrate that is used in the conjugate vaccine may besusceptible to aggregation upon storage and physical stresses.

As shown in FIG. 11 , the PCV16 and PCV21 drug products were stablethrough 4 weeks at 25° C. and 37° C. and up to 12 wks at 4° C.regardless of polysaccharide concentration. Moreover, upon horizontalagitation after thermal stress, the PCV16 and PCV21 drug products wereshown to be stable. No loss of antigen quantity due to non-specificadsorption to the side of the containers or vessels was observed underthese thermal and physical stresses. Additionally, no aggregation of thedrug product was observed which would impact its performance on thecolumn (e.g. irreversible binding to the column or unable to enter/exitthe resolving columns) in estimating total dose of the vaccine (FIG. 12).

HPSEC UV/MALs/RI was used to measure the molecular weight of apolysaccharide-protein conjugate. If the polysaccharide-proteinconjugates in the drug product were degrading or depolymerizing, adecrease in molecular weight would be evident and if aggregation of thedrug product was occurring, an increase in molecular weight would occur.This measurement provides additional characterization on the quality ofthe vaccine drug product or intermediate. As shown in FIG. 13 , thePCV16 and PCV21 drug products comprising drug substances that were eachprepared using reductive amination in an aprotic solvent (e.g. DMSO)were stable against depolymerization or chemical degradation of thecarbohydrate and stable against aggregation of the protein in the drugproduct formulation. This indicates that the PCV16 and PCV21 drugproducts using polysaccharide protein conjugates prepared usingreductive amination in an aprotic solvent, such as DMSO, are stablethrough elevated temperature and physical stresses. The formulationsdemonstrate excellent stability in terms of both quantity and quality.The formulation does not lose total dose due to non-specific adsorptionto the side of the containers or other surfaces and the formulation doesnot degrade or aggregate as indicated by the stable molecular weight ofthe vaccine drug product (weight averaged molecular weight (Mw) andnumber averaged molecular weight (Mn)).

Example 40

Impact of Conjugation Chemistry on Stability of a Pneumococcal ConjugateVaccine

Individual pneumococcal polysaccharide-protein conjugates preparedutilizing different chemistries as described in the Examples, supra,were used for the formulation of 15- and a 16-valent pneumococcalconjugate vaccines referred to as PCV15 and PCV16 at 64 μg/mL. The PCV15vaccine drug product contained serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 22F, 23F and 33F, conjugated using reductive amination ina protic solvent (all aqueous) and formulated in 20 mM L-Histidine pH5.8, 150 mM NaCl and 0.2% w/v PS-20 (see EXAMPLE 38). The PCV16 drugproduct contained serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F,17F, 20A, 23A, 23B, 24F, 31, and 35B, conjugated using reductiveamination in an aprotic solvent (e.g. all DMSO) and formulated in 20 mML-Histidine, pH 5.8, 150 mM NaCl, and 0.2% (w/v) PS-20 (see EXAMPLE 38).Each polysaccharide-protein conjugate was formulated at 4 μg/mL (w/v)Pneumococcal Polysaccharide (PnPs) except for 6B in PCV15, which wasformulated at 8 μg/mL, for a final concentration of 64 μg/mL PnPs ineach vaccine.

The vaccine-filled containers were placed at 4° C. or 37° C. for up to 1week. Intrinsic Protein Fluorescence Spectroscopy (IPFS) was used toassess the stability of pneumococcal polysaccharide-conjugated carrierprotein, CRM197, in the PCV15 or PCV16 compositions upon storage for upto 1 week at 4° C. and 37° C. Fluorescence emission spectra (Em 290-400nm) of undiluted samples were collected using a Jasco FP-6500Spectrofluorometer at ambient room temperature in a 1 cm path lengthquartz cuvette. An Excitation wavelength of 280 nm was used with a scanspeed of 100 nm/min and excitation and emission bandpass of 3 nm.

As shown in FIG. 14 , the drug product formulation containing drugsubstances prepared using reductive amination in an aprotic solvent(e.g. all DMSO) resulted in superior physical and chemical stability ascompared to a vaccine utilizing drug substances prepared using a proticsolvent during reductive amination in the conjugation process (allaqueous conjugation). In as little as 16 hours at 37° C., thefluorescence intensity of the PCV15 drug product, at 64 μg/mL PnPs,prepared using reductive amination in a protic solvent (all aqueous) forthe conjugation process was reduced and the emission maximum of thevaccine shifted from 332 nm to 338 nm. The PCV16 vaccine drug product,at 64 μg/mL PnPs, utilizing drug substances prepared with reductiveamination chemistry in an aprotic solvent (e.g. all DMSO) did not show achange in fluorescence intensity or emission maximum 338 nm. It shouldbe understood that the resulting signal intensity and emission maximumacquired from the IPFS measurement is due to the collective stability ofthe individual drug substances in a multivalent complex drug product. Wehave shown that a vaccine drug product utilizing all drug substancesprepared in an aprotic solvent is stable while a vaccine drug productutilizing all drug substances prepared in an a protic solvent is notstable.

Without being bound to any particular theory, this stability could bedue to conjugation in DMSO resulting in unfolding and denaturing thecarrier protein. Conjugation to the denatured carrier protein would lockthe conformation in denatured state post conjugation. If so, this studywould also suggest the final conformation is stable under the stressstudy evaluated here. It is therefore preferred to use a vaccine withconjugate mostly prepared with aprotic solvents to provide a consistentand stable vaccine drug product.

If a vaccine drug product composition included a mixture of drugsubstances, glycoconjugates or polysaccharide protein conjugatesutilizing different reductive amination solvents (aqueous or DMSO) inthe conjugation processes, the average emission maximum or intensitymeasured from the drug product formulation would be weighted due to thecontribution of each aqueous polysaccharide protein conjugate emissionat 332 nm and each non-aqueous polysaccharide protein conjugate emissionat 338 nm. It would be expected that a change would occur, in eitherintensity or emission maxima, upon elevated temperature for such amixture that does not use all polysaccharide protein conjugates,prepared using reductive amination in an aprotic solvent, in themultivalent drug product formulation. It is therefore preferred to use avaccine with conjugate mostly prepared with aprotic solvents to providea consistent and stable vaccine drug product.

Example 41

Compiled Monovalent Rabbit Immunogenicity Studies for Selected Serotypes

Adult New Zealand White rabbits (NZWR, n=3/group) were intramuscularly(IM) immunized with 0.25 ml or 0.5 mL (for 15C only) of respectivemonovalent conjugate vaccine on day 0 and day 14 (alternating sides).Monovalent pneumococcal vaccines, formulated in 20 mM L-Histidine, pH5.8, 150 mM NaCl, and 0.2% (w/v) PS-20 and formulation process describedin Example 38, were dosed as follows: (1) 1 μg PnPs (6C, 10A, 15A, 16F,17F, 20A, 23A, 23B, 24F, 31 or 35B, each conjugated to CRM197) with 62.5μg aluminum phosphate adjuvant (APA) per immunization, or (2) 2 μg PnPs(15C-CRM197 with 125 μg APA per immunization. Sera were collected priorto study start (pre-immune) and on days 14 (post-dose 1, PD1) and 28(post-dose 2, PD2). NZWRs were observed at least daily by trained animalcare staff for any signs of illness or distress. The vaccineformulations in NZWRs were deemed to be safe and well tolerated, as novaccine-related adverse events were noted. All animal experiments wereperformed in strict accordance with the recommendations in the Guide forCare and Use of Laboratory Animals of the National Institutes of Health.The NZWR experimental protocol was approved by the Institutional AnimalCare and Use Committees at both Merck & Co., Inc. (Kenilworth, NJ) andCovance (Denver, PA).

NZWR sera were tested in ELISA assays to evaluate IgG immunogenicityusing a 1-2 mg/mL respective PnPs coating concentration. Functionalantibody was determined through opsonophagocytosis assays (OPA) based onpreviously described protocols available online at the BacterialRespiratory Pathogen Reference Laboratory at the University of Alabamaat Birmingham using Opsotiter® 3 software (UAB Research Foundation,Caro-Aguilar et al. Immunogenicity differences of a 15-valentpneumococcal polysaccharide conjugate vaccine (PCV15) based on vaccinedose, route of immunization and mouse strain. Vaccine 35(6):865-72(2017); Burton et al. Development and validation of a fourfoldmultiplexed opsonization assay (MOPA4) for pneumococcal antibodies. ClinVaccine Immunol 13(9):1004-9 (2006)).

All monovalent pneumococcal conjugate vaccines were found to beimmunogenic in rabbits (FIG. 15 ) and generate functional antibody whichkilled the respective bacterial strain (FIG. 16 ).

Example 42

Evaluation of Cross Protection of Serotypes 15A, 15B, 15C

Rabbits were immunized with 15A-CRM197/APA, 15B-CRM197/APA or15C-CRM197/APA to evaluate cross-reactivity between each serotype.

Adult New Zealand White rabbits (NZWR, n=3/group) were intramuscularly(IM) immunized with 0.5 ml of respective monovalent conjugate vaccine onday 0 and day 14 (alternating sides). Monovalent pneumococcal conjugatevaccine, formulated in 20 mM L-Histidine, pH 5.8, 150 mM NaCl, and 0.2%(w/v) PS-20 and formulation process described in Example 38, was dosedat 2 μg PnPs (15A, 15B or 15C each conjugated to CRM197) with 125 μg APAper immunization. Sera were collected prior to study start (pre-immune)and on days 14 (post-dose 1, PD1) and 28 (post-dose 2, PD2). NZWRs wereobserved at least daily by trained animal care staff for any signs ofillness or distress. The vaccine formulations in NZWRs were deemed to besafe and well tolerated, as no vaccine-related adverse events werenoted. All animal experiments were performed in strict accordance withthe recommendations in the Guide for Care and Use of Laboratory Animalsof the National Institutes of Health. The NZWR experimental protocol wasapproved by the Institutional Animal Care and Use Committees at bothMerck & Co., Inc and Covance (Denver, PA).

NZWR sera were tested in ELISA assays to evaluate IgG immunogenicityusing a 1-2 mg/mL respective PnPs coating concentration. Functionalantibody was determined through OPA based on previously describedprotocols available online at the Bacterial Respiratory PathogenReference Laboratory at the University of Alabama at Birmingham usingOpsotiter® 3 software (UAB Research Foundation (Caro-Aguilar et al.,2017; Burton et al., 2006).

All three monovalent pneumococcal conjugate vaccines of serogroup 15were found to be immunogenic in rabbits (FIG. 17 ) and generatefunctional antibody which killed the respective bacterial strain (FIG.18 ). In addition, rabbits immunized with serogroup 15 monovalentpneumococcal conjugate vaccines had equivalent PD2 IgG and OPA titers tothe homologous and heterologous polysaccharide and bacterial strain,respectively. Rabbits immunized with 15A-CRM197/APA, 15B-CRM197/APA or15C-CRM197/APA all had cross reactivity to each pneumococcalpolysaccharide (15A, 15B, 15C) (FIG. 17 ). Using post dose 2 (PD2) logtransformed IgG data analyzed by One-way ANOVA with Tukey's multiplecomparison test (P-value=0.354), there was no significant difference inthe IgG titers across serogroup 15. In addition, rabbits immunized with15A-CRM197/APA, 15B-CRM197/APA or 15C-CRM197/APA all had crossreactivity to each S. pneumoniae bacterial strain, (15A, 15B, 15C), asall rabbit hyper immune sera had functional antibody to each strainevaluated and killed the bacteria (FIG. 18 ). Similarly, using post dose2 (PD2) log transformed OPA data analyzed by One-way ANOVA with Tukey'smultiple comparison test (P-Value=0.054), there was no significantdifference in OPA titers across the serogroup 15.

Example 43

Immunogenicity of PCV21 in New Zealand White Rabbits

Adult New Zealand White rabbits (NZWR, n=5/group) were intramuscularly(IM) immunized with 0.1-0.5 mL of pneumococcal conjugate vaccine on day0 and day 14 (alternating sides). A PCV21 pneumococcal vaccine,formulated in 20 mM L-Histidine, pH 5.8, 150 mM NaCl, and 0.2% (w/v)PS-20 and formulation process described in Example 38, was dosed at 4,2, 1, 0.4, 0.08 or 0.016 μg PnPs (3, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A,15C, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F or 35B, eachconjugated to CRM197) per immunization. Sera were collected prior tostudy start (pre-immune) and on days 14 (post-dose 1, PD1) and 28(post-dose 2, PD2). NZWRs were observed at least daily by trained animalcare staff for any signs of illness or distress. The vaccineformulations in NZWRs were deemed to be safe and well tolerated, as novaccine-related adverse events were noted. All animal experiments wereperformed in strict accordance with the recommendations in the Guide forCare and Use of Laboratory Animals of the National Institutes of Health.The NZWR experimental protocol was approved by the Institutional AnimalCare and Use Committees at both Merck & Co., Inc and Covance (Denver,PA).

NZWR sera were evaluated for IgG immunogenicity using a multiplexedelectrochemiluminescence (ECL) assay. This assay was developed for usewith rabbit serum based on the human assay described by Marchese et al.(Optimization and validation of a multiplex,electrochemiluminescence-based detection assay for the quantitation ofimmunoglobulin G serotype-specific antipneumococcal antibodies in humanserum. Clin Vaccine Immunol. 16(3): 387-96 (2009)) using technologydeveloped by MesoScale Discovery (a division of MesoScale Diagnostics,LLC, Gaithersburg, MD) which utilizes a SULFO-TAG™ label that emitslight upon electrochemical stimulation. SULFO-TAG™-labeled anti-rabbitIgG was used as the secondary antibody for testing NZWR serum samples.Functional antibody was determined through multiplexed opsonophagocyticassays (MOPA) based on previously described protocols available onlineat the Bacterial Respiratory Pathogen Reference Laboratory at theUniversity of Alabama at Birmingham using Opsotiter® 3 software (UABResearch Foundation, Caro-Aguilar et al, 2017, supra, Burton et al.,2006, supra).

PCV21 pneumococcal conjugate vaccines were found to be immunogenic inrabbits (FIGS. 19A, 19B, 20A, and 20B) and generate functional antibodywhich killed vaccine-type bacterial strains (FIGS. 21A and 21B) at alldoses tested.

Comparable PD1 immunogenicity was observed for PCV21 dosed at 4, 2, 1and 0.4 μg per PnPs, with the exception of 24F which had higherimmunogenicity in the 2 μg dose compared to the 4 or 1 μg doses and 15A,15B and 15C which had higher immunogenicity in the 0.4 μg dose comparedto the 2 μg dose (FIGS. 19A and 19B). Therefore, there was not much of avaccine dose response at the higher PnPs doses (0.4-4 μg/PnPs). Lowerimmunogenicity was observed at the 0.08 and 0.016 μg PnPs vaccine doses,as many serotypes were significantly lower when compared to the 2 μgvaccine dose.

Comparable PD2 immunogenicity was observed for PCV21 dosed at 4, 2, 1and 0.4 μg per PnPs (FIGS. 20A and 20B).

In general comparable PD2 MOPA titers were observed for PCV21 dosed at4, 2, 1 and 0.4 μg per PnPs with MOPA titers trending lower for the 0.08and 0.016 μg PnPs vaccine doses, although many not reaching statisticalsignificance (data not shown). PCV21 dosed at 2 μg per PnPs was selectedas a representative data set for MOPA. Specifically, rabbits immunizedwith PCV21 at the 2 μg dose had significantly higher PD1 MOPA titers forall serotypes compared to pre-immune rabbit sera with the exception ofserotype 3 (FIG. 21A). It should be noted that rabbit pre-immune serafrom this study had higher background titers to serotypes 16F, 31 and35B. Rabbits immunized with PCV21 at the 2 μg dose had significantlyhigher PD2 MOPA titers for all serotypes compared to pre-immune rabbitsera (FIG. 21B). Log Transformed data were analyzed by One-way ANOVAwith Dunnett's test to determine significance.

Example 44

Materials and Methods

Free Polysaccharide Testing

Free polysaccharide (polysaccharide that is not conjugated with CRM197)in conjugate sample is measured by first precipitating free protein andconjugates with deoxycholate (DOC) and hydrochloric acid. Precipitatesare then filtered out and the filtrates are analyzed for freepolysaccharide concentration by HPSEC/UV/MALS/RI. Free polysaccharide iscalculated as a percentage of total polysaccharide measured byHPSEC/UV/MALS/RI.

Free Protein Testing

Free polysaccharide, polysaccharide-CRM197 conjugate, and free CRM197 inconjugate samples are separated by capillary electrophoresis in micellarelectrokinetic chromatography (MEKC) mode. Briefly, samples are mixedwith MEKC running buffer containing 25 mM borate, 100 mM SDS, pH 9.3,and are separated in a preconditioned bare-fused silica capillary.Separation is monitored at 200 nm and free CRM197 is quantified with aCRM197 standard curve. Free protein results are reported as a percentageof total protein content determined by the HPSEC/UV/MALS/RI procedure.

Molecular Weight and Concentration Analysis of Conjugates Using HPSEC UVMALS/RI Assay

Conjugate samples were injected and separated by high performancesize-exclusion chromatography (HPSEC). Detection was accomplished withultraviolet (UV), multi-angle light scattering (MALS) and refractiveindex (RI) detectors in series. Protein concentration was calculatedfrom UV280 using an extinction coefficient. Polysaccharide concentrationwas deconvoluted from the RI signal (contributed by both protein andpolysaccharide) using the dn/dc factors which are the change in asolution's refractive index with a change in the solute concentrationreported in mL/g. Average molecular weight of the samples werecalculated by Astra software (Wyatt Technology Corporation, SantaBarbara, CA) using the measured concentration and light scatteringinformation across the entire sample peak. There are multiple form ofaverage values of molecular weight for polydispersed molecules. Forexample number-average molecular weight Mn, weight-average molecularweight Mw, and z-average molecular weight Mz (Molecules, 2015, 20,10313-10341). Unless specified, the molecular weights are weight-averagemolecular weight.

Determination of Lysine Consumption in Conjugated Protein as a Measureof the Number of Covalent Attachments Between Polysaccharide and CarrierProtein

The Waters AccQ-Tag amino acid analysis (AAA) was used to measure theextent of conjugation in conjugate samples. Samples were hydrolyzedusing vapor phase acid hydrolysis in the Eldex workstation, to break thecarrier proteins down into their component amino acids. The free aminoacids were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC). The derivatized samples were then analyzed using UPLCwith UV detection on a C18 column. The average protein concentration wasobtained using representative amino acids other than lysine. Lysineconsumption during conjugation (i.e., lysine loss) was determined by thedifference between the average measured amount of lysine in theconjugate and the expected amount of lysine in the starting protein.

Example 45

Impact of Conjugation Process on Stability of a Pneumococcal ConjugateVaccine

Individual pneumococcal polysaccharide-protein conjugates preparedutilizing different reductive amination solvents (aqueous or DMSO) asdescribed in the Examples, supra, were used for the formulation of a 1-,7-, 14-, and 21-valent pneumococcal conjugate vaccine drug productreferred to as PCV1, PCV7, PCV14 and PCV21 at 84 μg/mL.

The drug product vaccine-filled containers were placed at 4° C. or 37°C. for up to 7 days. Intrinsic Protein Fluorescence Spectroscopy (IPFS)was used to assess the stability of pneumococcalpolysaccharide-conjugated carrier protein, CRM197, in the PCV1, PCV7,PCV14 or PCV21 compositions upon storage for up to at 4° C. and 37° C.Fluorescence emission spectra (Em 290-400 nm) of undiluted samples werecollected using a Jasco FP-6500 Spectrofluorometer at ambient roomtemperature in a 1 cm path length quartz cuvette. An Excitationwavelength of 280 nm was used with a scan speed of 100 nm/min andexcitation and emission bandpass of 3 nm.

As shown in FIGS. 22 A-C, the drug product formulations containing allpolysaccharide-protein conjugates prepared using reductive amination inan aprotic solvent (e.g. DMSO) resulted in superior physical andchemical stability as compared to a vaccine utilizing allpolysaccharide-protein conjugates prepared using aqueous solvent duringreductive amination in the conjugation process (protic). In as little as16 hours at 37° C., the fluorescence intensity of the PCV1, PCV7 andPCV14 drug products prepared using all polysaccaride-protein conjugateswhich were prepared using reductive amination in an aqueous solvent at84 μg/mL PnPs was reduced and the emission maximum of the vaccineshifted from 332 nm to 338 nm. The PCV1, PCV7 and PCV14 vaccine drugproducts, at 84 μg/mL PnPs, utilizing drug substances prepared withreductive amination chemistry in an aprotic solvent (e.g. DMSO) did notshow a change in fluorescence intensity or emission maximum at 338 nm.Moreover, a PCV21 drug product was also studied. It was formulated in 20mM L-Histidine, pH 5.8, 150 mM NaCl, and 0.1% (w/v) PS-20 as describedin Example 38, containing polysaccharide-protein conjugates preparedusing reductive amination in an aprotic solvent (e.g. DMSO). The studyshowed superior physical and chemical stability after storage at 37° C.PCV21 did not show a change in fluorescence intensity or emissionmaximum 338 nm (FIG. 22D).

It should be understood that the resulting signal intensity and emissionmaximum acquired from the IPFS (EXAMPLE 40) measurement is due to thecollective stability of the individual polysaccharide-protein conjugatesin a multivalent complex drug product. We have shown that a vaccine drugproduct utilizing all polysaccharide-protein conjugates prepared in anaprotic solvent is stable while a vaccine drug product utilizing alldrug substances prepared in a protic solvent is not stable understressed conditions. If a vaccine drug product composition included amixture of polysaccharide-protein conjugates using both protic and anaprotic conjugation process, the average emission maximum or intensitymeasured from the drug product would be weighted due to the contributionof each protic polysaccharide-protein conjugates emission at 332 nm andeach aprotic of polysaccharide-protein conjugate emission at 338 nm. Itwould be expected that a weighted change would occur, in eitherintensity or emission maxima, upon elevated temperature for such amixture due to the presence of the polysaccharide protein conjugatesprepared using a protic conjugation process.

As shown in FIG. 23 , to assess stability of the PCV21 drug productprepared at 0.084 mg/mL PnPs, Nanoparticle Tracking Analysis (NTA) wasutilized. The technique collects videos of directly tracked nanoparticlepopulations as they move by Brownian motion to extrapolate particle sizeand concentration. A class 1, 635 nm laser focuses an 80 micron redlaser beam through the liquid sample, illuminating particles as rapidlydiffusing points of light. A CCD camera records a 30 frame per secondvideo to track the movement of each individual illuminated particle overtime. The system software identifies the center of each individualparticle from the video and tracks the distance independently traversedto determine the mean square displacement. This tracking is performedsimultaneously for every particle within the sample population in eachframe until the raw data collected from the entire video is analyzed. Bysimultaneously measuring the mean square displacement of everyindividual particle tracked, its diffusion coefficient (Dt) and thespherical equivalent hydrodynamic radius (rh) are determined by applyingthe Stokes-Einstein equation. The software then represents thisaccumulated data as a particle size and concentration distribution. Rawdata information on not only particle size and concentration, but alsointensity, or brightness of the individual particle are gathered. Takentogether the data are fitted and plotted individually as particleintensity relative to particle size, and particle concentration relativeto particle size, and then on three dimensional contour plots comparingparticle size, concentration, and intensity of all particle populations.

A conjugate vaccine comprised of carrier protein and polysaccharideantigen may together or separately be susceptible to aggregation withinthe 10-1000 nm particle size range, thereby making NTA a suitablestability indicating technique for assessing and quantitatingaggregation phenomena. Upon exposure of drug product to 4° C. and 37° C.for 1 week, no significant aggregation of drug product was observed byNTA for formulations containing PS-20 at concentrations of 0.025% w/v,0.05% w/v, 0.1% w/v, 0.15% w/v, and 0.2% w/v PS20 (as shown in FIG. 23). However, after subjecting formulations to agitation for 6 hr. at 4°C., the formulation lacking polysorbate 20 was ineffective at mitigatingagitation induced aggregation of the drug product as evidenced byincreased D90 particle size, broadened particle size distribution, andincreased particle intensity as provided by NTA. Therefore it ispreferred to keep polysorbate 20 at 0.025 to 0.2% or higher levels (w/v)to stabilize the drug product during routine manufacturing, storage,shipping and handling.

As described in Example 39, HPSEC UV/MALS/RI was used to measure themolecular weight of a 21-valent (PCV21) polysaccharide-protein conjugatedrug product, formulated in 20 mM L-Histidine, pH 5.8, 150 mM NaCl, and0 to 0.2% (w/v) PS-20 and formulation process described in Example 38.The formulations were placed at 4° C. or 37° C. for up to one week. Insome cases, the syringes were placed on a horizontal rotation platformand shaken for up to 6 hours after storage for 1 week at 4° C. or 37° C.This was done to simulate the shipping and handling stresses that mayaccompany drug product manufacture and distribution. If thepolysaccharide-protein conjugates in the drug products were degrading ordepolymerizing, a decrease in molecular weight would be evident and ifaggregation of the drug product formulation was occurring, an increasein molecular weight would occur. This measurement provides additionalcharacterization on the quality of the vaccine drug product orpolysaccharide-protein conjugates as a function of PS-20 concentration.As shown in FIG. 24 , the PCV21 drug product formulations comprising alldrug substances that were each prepared using reductive amination in anaprotic solvent (e.g. DMSO) were stable against depolymerization orchemical degradation of the carbohydrate and stable against aggregationof the protein in the drug product formulation due to heat treatmentbetween 0.05% and 0.15% PS-20. Collectively, this indicates that a PCV21drug product is stable through elevated temperature and physicalstresses when formulated using PS-20 from 0.025% and 0.2% or higherlevels.

Example 46

PCV21 Protection from Challenge in Mice

Young female Swiss Webster mice (6-8 weeks old, n=10/group) wereintraperitoneally (IP) immunized with 0.1-0.5 mL of a 21-valentpneumococcal conjugate vaccine (PCV21) on days 0, day 14, and day 28.PCV21 pneumococcal vaccine was dosed at 4, 2, 0.4, 0.08 or 0.016 μg PnPs(3, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20A, 22F,23A, 23B, 24F, 31, 33F and 35B each conjugated to CRM197) perimmunization. Mice were observed at least daily by trained animal carestaff for any signs of illness or distress. The vaccine formulations inmice were deemed to be safe and well tolerated, as no vaccine-relatedadverse events were noted. On day 52 the mice were intratracheallychallenged with Streptococcus pneumoniae serotype 24F. Exponential phasecultures of S. pneumoniae were centrifuged, washed, and suspended insterile PBS. Mice were anesthetized with isoflurane prior to challenge.105 cfu of S. pneumoniae in 0.1 mL of PBS was placed in the throat ofmice hung upright by their incisors. Aspiration of the bacteria wasinduced by gently pulling the tongue outward and covering the nostrils.Mice were weighed daily and euthanized if weight loss exceeded 20% ofstarting weight. Blood was collected at 24 hours, 48 hours, and 72 hoursto assess for bacteremia. Mice were observed at least twice daily bytrained animal care staff for any signs of illness or distress. Allanimal experiments were performed in strict accordance with therecommendations in the Guide for Care and Use of Laboratory Animals ofthe National Institutes of Health. The mouse experimental protocol wasapproved by the Institutional Animal Care and Use Committee at Merck &Co., Inc.

Mouse sera were evaluated for IgG immunogenicity using a multiplexedelectrochemiluminescence (ECL) assay. This assay was developed for usewith mouse serum based on the human assay described by Marchese et al.,Clin Vaccine Immunol. (2009) 16(3):387-96, using technology developed byMesoScale Discovery (a division of MesoScale Diagnostics, LLC,Gaithersburg, MD) which utilizes a SULFO-TAG™ label that emits lightupon electrochemical stimulation. SULFO-TAG™-labeled anti-mouse IgG wasused as the secondary antibody for testing mouse serum samples.Functional antibody was determined through multiplexed opsonophagocyticassays (MOPA) based on previously described protocols atwww.vaccine.uab.edu and Opsotiter® 3 software owned by and licensed fromUniversity of Alabama (UAB) Research Foundation (See, Caro-Aguilar I. etal., Vaccine (2017) 35(6):865-72 and Burton R. L. and Nahm M. H. Clin.Vaccine Immunol. (2006) 13(9):1004-9).

PCV21 immunization generated antibody titers in Swiss Webster mice forall serotypes in the vaccine (data not shown). PCV21 was alsoimmunogenic in Balb/c and CD1 mice (data not shown). PCV21 immunizedSwiss Webster mice were also protected from challenge with S. pneumoniaeserotype 24F (FIG. 25 ). Mantel Cox log-rank test indicates that allPCV21 immunized groups were significantly protected from challenge whencompared to the naïve group (P<0.05). Likewise, PCV21 immunized mousegroups had little to no bacteremia, which was significantly less whencompared to the naïve group (data not shown).

Example 47

PCV Immunogenicity and Functional Antibody in Rabbits

Adult New Zealand white rabbits (NZWR, n=5/group) were intramuscularly(IM) immunized with 0.1 mL of PCV on day 0 and day 14 (alternatingsides). PCV was dosed at 0.4 μg PnPs per serotype per immunization with25 μg [Al] in the form of Aluminum Phosphate Adjuvant (APA). In the caseof PCV31, the dosing concentration of serotype 6B was doubled to 0.8 μgPnPs per immunization. PCV21 (as described in EXAMPLE 38) includedpurified polysaccharides from serotypes 3, 6C, 7F, 8, 9N, 10A, 11A, 12F,15A, 15C, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F and 35B eachconjugated to CRM197. Each polysaccharide-protein conjugate was preparedunadjuvanted or formulated with 250 μg [Al]/mL in the form of AluminumPhosphate or APA to evaluate the effect of adjuvant (PCV21/APA). Lowervalent PCVs were also formulated (as described in EXAMPLE 38) to include8 novel STs (PCV8: 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B eachconjugated to CRM197, unadjuvanted), the 16 STs not included in anylicensed PCV (PCV16: 6C, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 20,23A, 23B, 24F, 31 and 35B each conjugated to CRM197, unadjuvanted) orPCV31 (1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A,15C, 16F, 17F, 18C, 19A, 19F, 20A, 22F, 23A, 23B, 23F, 24F, 33F and 35Beach conjugated to CRM197, formulated with 250 μg [Al]/mL in the form ofAluminum Phosphate) to evaluate carrier suppression as vaccine valencyincreased (PCV31/APA). Each vaccine used polysaccharide proteinconjugates formulated at 4 μg/mL (w/v) Pneumococcal Polysaccharide(PnPs), except for 6B which was formulated at 8 μg/mL (w/v) PneumococcalPolysaccharide (PnPs) in PCV31 or PCV31/APA. The final concentration ofPneumococcal Polysaccharide (PnPs) in each vaccine was 128 μg/mL PnPs inPCV31, 84 μg/mL PnPs in PCV21, 64 μg/mL in PCV16 and 32 μg/mL in PCV8.

Sera were collected prior to study start (pre-immune) and on days 14(PD1) and 28 (PD2). NZWRs were observed at least daily by trained animalcare staff for any signs of illness or distress. The vaccineformulations in NZWRs were deemed to be safe and well tolerated, as novaccine-related adverse events were noted. All animal experiments wereperformed in strict accordance with the recommendations in the Guide forCare and Use of Laboratory Animals of the National Institutes of Health.The NZWR experimental protocol was approved by the Institutional AnimalCare and Use Committees at both Merck & Co., Inc and Covance (Denver,PA).

Rabbit sera were evaluated for IgG immunogenicity using a multiplexedelectrochemiluminescence (ECL) assay. This assay was developed for usewith rabbit serum based on the human assay described by Marchese et al.,Clin Vaccine Immunol. (2009) 16(3):387-96, using technology developed byMesoScale Discovery (a division of MesoScale Diagnostics, LLC,Gaithersburg, MD) which utilizes a SULFO-TAG™ label that emits lightupon electrochemical stimulation. SULFO-TAG™-labeled anti-rabbit IgG wasused as the secondary antibody for testing NZWR serum samples.Functional antibody was determined through multiplexed opsonophagocyticassays (MOPA) based on previously described protocols atwww.vaccine.uab.edu and Opsotiter® 3 software owned by and licensed fromUniversity of Alabama (UAB) Research Foundation (See, Caro-Aguilar I. etal., Vaccine (2017) 35(6):865-72 and Burton R. L. and Nahm M. H. Clin.Vaccine Immunol. (2006) 13(9):1004-9).

Rabbit sera were tested as a pool for pre-immune and PD1, andindividually for PD2 in multiplexed electrochemiluminescent assays todetermine antibody titers. PCVs generated antibody titers in rabbits forall serotypes following immunizations with the vaccine (data not shown).Serotype 15B polysaccharide was not included in PCV16, PCV21 or PCV31,however antibody titers to serotype 15B were observed followingimmunization with both PCV16, PCV21 and PCV31.

PCV21 without adjuvant had comparable to or significantly higherimmunogenicity (serotypes 3, 7F, 10A, 19A, 23A and 23B) when compared toPCV21 with APA (FIG. 26A-B and FIG. 28 ), suggesting no additionalimmunogenicity benefit in rabbits with APA included in the vaccine.

Carrier induced epitopic suppression refers to interference with theantibody response to an antigen (such as capsular polysaccharide)coupled to the same carrier protein (such as CRM197). Interference isthought to arise from competition for a limited number of carrierspecific primed T helper cells. As a result, there may be a decrease inresponse to the capsular polysaccharide. Pfizer has observed a decreasein vaccine immunogenicity of shared serotypes as the vaccine valencyincreased from a 7-valent to 13-valent [Comparison of IgG antibody GMCof Prevnar (7 valent) vs Prevnar13. (table 9, page 29 of PCV13monograph)]. Therefore, we sought to investigate the immunogenicity oflower valent PCVs compared to PCV21 (FIG. 27A-C). PCV8 (8 novelserotypes) had comparable immunogenicity relative to PCV21 for the 8shared serotypes (FIG. 29 ). PCV16 (PCV21 minus 5 overlapping serotypesfrom PCV15) had comparable immunogenicity relative to PCV21 for 15 ofthe 16 shared serotypes (FIG. 29 ). Serotype 31 had higherimmunogenicity in PCV16 (FIG. 29 ). Overall, there were no major trendsof carrier suppression with PCV21 in NZWR. PCV31/APA had comparableimmunogenicity relative to PCV21/APA for the 12 of the 21 sharedserotypes. 10 serotypes (6C, 7F, 9N, 12F, 15A, 15B (not in PCV21), 15C,17F, 19A, and 23A) had higher immunogenicity in PCV31, ranged between2.9-fold and 11-fold (FIG. 30 ). Overall, there were no major trends ofcarrier suppression with PCV31 in NZWR.

PCVs were found to be immunogenic in rabbits and generated functionalantibodies which killed vaccine-type bacterial strains. Rabbit sera weretested in multiplexed opsonophagocytic assays (MOPA) to determinefunctional antibody titers. PCV21 had comparable to or higher PD2 OPAtiters when compared to PCV21/APA, PCV8 and PCV16 (FIG. 31 ). LogTransformed data were analyzed by One-way ANOVA with Dunnett's test todetermine significance. PCV21 had significantly higher OPA titers forserotype 16F compared to PCV8 and for serotypes 12F, 23A and 19Acompared to PCV21/APA. PCV31 had significantly higher OPA titers forserotype 23A compared to PCV21.

Example 48

PCV21 Immunogenicity in Adult Rhesus Macaques

PCV21 was also assessed in adult Rhesus macaque immunogenicity models.Rhesus macaques were intramuscularly immunized with PCV21 on days 0, 28and 56. PCV21 was dosed at 1 μg PnPs in a 0.25 mL volume (3, 6C, 7F, 8,9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31,33F and 35B each conjugated to CRM197) per immunization. Sera werecollected prior to study start (pre-immune, day 0) and on days 14 (PD1),28, 42 (PD2), 56, 70 (PD3) and 84.

Rhesus sera were evaluated for IgG immunogenicity using a multiplexedelectrochemiluminescence (ECL) assay. This assay was developed for usewith Rhesus serum based on the human assay described by Marchese et al.and Skinner et al. (Marchese R. D. et al., Clin. Vaccine Immunol. (2009)16(3):387-96 and Skinner, J. M. et al., Vaccine (2011) 29(48):8870-8876)using technology developed by MesoScale Discovery (a division ofMesoScale Diagnostics, LLC, Gaithersburg, MD) which utilizes aSULFO-TAG™ label that emits light upon electrochemical stimulation.SULFO-TAG™-labeled anti-human IgG was used as the secondary antibody fortesting Rhesus serum samples. Functional antibody was determined throughmultiplexed opsonophagocytic assays (MOPA) based on previously describedprotocols at www.vaccine.uab.edu and Opsotiter® 3 software owned by andlicensed from University of Alabama (UAB) Research Foundation (See,Caro-Aguilar I. et al., Vaccine (2017) 35(6):865-72 and Burton R. L. andNahm M. H. Clin. Vaccine Immunol. (2006) 13(9):1004-9).

PCV21 was found to be immunogenic in adult monkeys and generatedfunctional antibodies which killed vaccine-type bacterial strains at alltime points tested (FIGS. 32 and 33 ). It is also of note that PCV21,which contains polysaccharide conjugates 15A-CRM197 and 15C-CRM197, alsoprovides cross-reactivity to 15B, as evidenced in ECL. PCV21 wasimmunogenic with one dose of vaccine in the monkeys (FIG. 32 ). Mostserotypes reached their maximum titer at PD1, with the exception of STs6C, 12F and 24F, which benefited from additional immunizations. Theadult Rhesus monkeys had higher pre-existing antibody titers as comparedto former PCV studies in infant Rhesus monkeys. The pre-existingantibody titers were most apparent when testing the sera samples inMOPA, which made determining the opsonophagocytic titer difficult. Allstudy time points were tested in MOPA as pooled or individual samples,but for easier viewing, only pre-immune, PD1 and PD3 are shown (FIG. 33), as there was not much difference in the OPA titers between PD1 andPD3.

PCV21 immunized adult Rhesus macaque sera were evaluated for crossprotection to other S. pneumoniae bacteria (FIG. 34 ). PCV21 immunizedmacaque sera had cross protection with serotypes 6A, 6B and 23F but not19F. The cross protection to 6A and 6B is likely due to immunizationwith polysaccharide conjugate 6C-CRM197 as part of a multivalent PCV21.Similarly, immunization with polysaccharide conjugates 23A-CRM197 and23B-CRM197 as part of a multivalent PCV resulted in cross protection toserotype 23F. Immunization with PCV21 including polysaccharide conjugate19A-CRM197 did not provide cross protection to 19F.

Example 49

PCV21 Immunogenicity in Adult Rhesus Macaques—Evaluation of Adjuvant andCarrier Suppression

Another monkey study included evaluation of PCV21 with and without APAand comparison of shared serotype immunogenicity to evaluate carriersuppression. Adult Rhesus macaques (n=5/group) were intramuscularlyimmunized with vaccines at a half human dose on day 0 of the study.PCV21 was dosed at 1 μg PnPs in a 0.25 mL volume (3, 6C, 7F, 8, 9N, 10A,11A, 12F, 15A, 15C, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F and35B each conjugated to CRM197) per immunization. One group includedPCV21, adjuvanted with 62.5 μg [Al] in the form of Aluminum Phosphate.Another group included PCV15 dosed at 1 μg PnPs in a 0.25 mL volume (1,3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F each conjugatedto CRM197 with 6B dosed at 2 μg) per immunization and adjuvanted with62.5 μg [Al] in the form of Aluminum Phosphate. Another group includedPrevnar13® (PCV13) dosed at 1.1 μg PnPs in a 0.25 mL volume (1, 3, 4, 5,6A, 7F, 9V, 14, 18C, 19A, 19F and 23F each conjugated to CRM197 with 6Bdosed at 2.2 μg) per immunization. Sera were collected prior to studystart (pre-immune, day 0) and on day 28.

PCV21 was found to be immunogenic in adult monkeys and generatedfunctional antibodies which killed vaccine-type bacterial strains. PCV21without adjuvant had comparable to or significantly higherimmunogenicity (serotypes 15A and 15B) when compared to PCV21 with APA(FIG. 35 ). PCV21 did not include serotype 15B.

PCV21 immunized monkeys were compared to those immunized with PCV15 andPrevnar13. PCV21 and PCV15 have 5 shared serotypes (3, 7F, 19A, 22F,33F); there was no difference in immunogenicity of those 5 serotypesbetween monkeys immunized with PCV21 or PCV15 (FIG. 36 ). PCV21 andPrevnar13 have 3 shared serotypes (3, 7F, 19A); there was no differencein immunogenicity for serotypes 7F and 19A. Prevnar13 immunized monkeyshad lower antibody titers for serotype 3 compared to PCV21 (FIG. 36 ).This finding is consistent with the PCV15 human clinical data, showingserotype 3 to be more immunogenic in Merck's PCV15 vaccine. Takentogether, these results suggest that carrier suppression was notobserved in adult Rhesus macaques immunized with PCV21 when compared toPrevnar13 or PCV15.

Example 50

Serotype 6A, 6B and 6C Cross Reactivity—Monovalent Study

The monovalent drug product was prepared using Pneumococcalpolysaccharide 6A-CRM197 conjugate or Pneumococcal polysaccharide6B-CRM197 conjugate and was formulated in 20 mM histidine pH 5.8 and 150mM sodium chloride and 0.1% w/v polysorbate-20 (PS-20) at a target totalpolysaccharide concentration of 4.0 μg/mL of either serotype. Theconjugates were prepared by individually conjugating the CRM197 proteinto Pneumococcal polysaccharide (PnPs) types (either -6A or -6B). Therequired volume of bulk conjugates needed to obtain the targetconcentration of individual serotypes were calculated based on batchvolume and concentration of individual bulk polysaccharideconcentrations. The individual conjugates were added to a solution ofhistidine, sodium chloride and PS-20 to create a 2× conjugate blend. Theformulation vessel containing the 2× conjugate blend were mixed using amagnetic stir bar, and sterile filtered into another vessel. The sterilefiltered 2× blend was then diluted with saline to achieve the desiredtarget total polysaccharide and excipient concentrations. Theformulations were then filled into vials and stored at 2-8° C.

Rabbits were immunized with 6A-CRM197 or 6B-CRM197 to evaluatecross-reactivity within serogroup 6. Adult New Zealand White rabbits(NZWR, n=3/group) were intramuscularly (IM) immunized with 0.25 mL ofrespective monovalent conjugate vaccine on day 0 and day 14 (alternatingsides). Monovalent pneumococcal conjugate vaccine, formulated in 20 mML-Histidine pH 5.8 150 mM NaCl and 0.1% (w/v) PS-20 with a formulationprocess described in Example 38, was dosed at 1 μg PnPs (6A or 6B eachconjugated to CRM197). Sera were collected prior to study start(pre-immune) and on days 14 (post-dose 1, PD1) and 28 (post-dose 2,PD2). NZWRs were observed at least daily by trained animal care stafffor any signs of illness or distress. The vaccine formulations in NZWRswere deemed to be safe and well tolerated, as no vaccine-related adverseevents were noted. All animal experiments were performed in strictaccordance with the recommendations in the Guide for Care and Use ofLaboratory Animals of the National Institutes of Health. The NZWRexperimental protocol was approved by the Institutional Animal Care andUse Committees at both Merck & Co., Inc and Covance (Denver, PA).

NZWR sera were tested in ELISA assays to evaluate IgG immunogenicityusing a 2 μg/mL respective PnPs coating concentration. Functionalantibody was determined through opsonophagocytosis assays (OPA) based onpreviously described protocols at www.vaccine.uab.edu and Opsotiter® 3software owned by and licensed from UAB Research Foundation (See,Caro-Aguilar I. et al., Vaccine (2017) 35(6):865-72 and Burton R. L. andNahm M. H. Clin. Vaccine Immunol. (2006) 13(9):1004-9).

Both 6A-CRM197 and 6B-CRM197 were found to be immunogenic in rabbits(FIG. 37 ) and generate functional antibody which killed the respectivebacterial strain (FIG. 38 ). In addition, rabbits immunized withserogroup 6 monovalent pneumococcal conjugate vaccines had equivalentPD2 IgG and OPA titers to the homologous and heterologous polysaccharideand bacterial strain, respectively. Rabbits immunized with 6A-CRM197 or6B-CRM197 all had cross reactivity to each pneumococcal polysaccharide(PnPs 6A, PnPs 6B and PnPs 6C) (FIG. 37 ). Using post-dose 2 (PD2) logtransformed IgG data analyzed by One-way ANOVA, there was no significantdifference in the IgG titers across serogroup 6. In addition, rabbitsimmunized with 6A-CRM197 or 6B-CRM197 all had cross reactivity to eachS. pneumoniae bacterial strain (6A, 6B and 6C), as all rabbit hyperimmune sera had functional antibody to each strain evaluated and killedthe bacteria (FIG. 38 ). Similarly, using post-dose 2 (PD2) logtransformed OPA data analyzed by One-way ANOVA, there was no significantdifference in OPA titers across the serogroup 6.

Example 51

Serotype 20A and 20B Cross Reactivity—Monovalent Study

The monovalent drug product was prepared using Pneumococcalpolysaccharide 20A-CRM197 conjugate and was formulated in 20 mMhistidine pH 5.8 and 150 mM sodium chloride and 0.2% w/v Polysorbate-20(PS-20) at 4.0 μg/mL. The formulation was prepared with 250.0 μg [Al]/mLin the form of Aluminum Phosphate as the adjuvant (20A-CRM197/APA). Theconjugate was prepared by individually conjugating the CRM197 protein toPneumococcal polysaccharide (PnPs) type 20. The required volume of bulkconjugate needed to obtain the target concentration of individualserotype was calculated based on batch volume and concentration ofindividual bulk polysaccharide concentration. The single conjugate wasadded to a solution of histidine, sodium chloride and PS-20 to produce a4× conjugate blend at 16.0 μg/mL. The formulation vessel containing theconjugate blend was mixed using a magnetic stir bar and the sterile wasfiltered into another vessel. The sterile filtered 4× blend was thenadded to another vessel containing Aluminum Phosphate Adjuvant toachieve the desired target total polysaccharide, excipient and adjuvantconcentrations. The formulations were then filled into glass vials andstored at 2-8° C.

Rabbits were immunized with 20A-CRM197/APA or PCV21 to evaluatecross-reactivity within serogroup 20. Adult New Zealand White rabbits(NZWR, n=3/group) were intramuscularly (IM) immunized with pneumococcalconjugate vaccine on day 0 and day 14 (alternating sides). Monovalentpneumococcal conjugate vaccine, formulated as described above, was dosedat 1 μg PnPs in 0.25 ml (ST20A conjugated to CRM197 and 62.5 μg [Al] inthe form of Aluminum Phosphate was used as the adjuvant), a PCV21multivalent pneumococcal conjugate vaccine (84 μg/mL PnPs), formulatedin 20 mM L-Histidine pH 5.8 150 mM NaCl and 0.2% (w/v) PS-20 with aformulation process described in Example 38, was dosed at 0.4 μg PnPs in0.1 ml (serotypes 3, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F,19A, 20, 22F, 23A, 23B, 24F, 31, 33F and 35B each conjugated to CRM197).Sera were collected prior to study start (pre-immune) and on days 14(post-dose 1, PD1) and 28 (post-dose 2, PD2). NZWRs were observed atleast daily by trained animal care staff for any signs of illness ordistress. The vaccine formulations in NZWRs were deemed to be safe andwell tolerated, as no vaccine-related adverse events were noted. Allanimal experiments were performed in strict accordance with therecommendations in the Guide for Care and Use of Laboratory Animals ofthe National Institutes of Health. The NZWR experimental protocol wasapproved by the Institutional Animal Care and Use Committees at bothMerck & Co., Inc and Covance (Denver, PA).

NZWR sera were tested in opsonophagocytosis assays (OPA) to evaluatefunctional antibody based on previously described protocols atwww.vaccine.uab.edu and Opsotiter® 3 software owned by and licensed fromUAB Research Foundation (See, Caro-Aguilar I. et al., Vaccine (2017)35(6):865-72 and Burton R. L. and Nahm M. H. Clin. Vaccine Immunol.(2006) 13(9):1004-9).

Rabbits immunized with both monovalent 20A-CRM197/APA and multivalentPCV21 vaccines generated functional antibody which killed both S.pneumoniae serotypes 20A and 20B (FIG. 39 ). This suggests that serotype20A and 20B share structural similarity, resulting in cross-reactivitywithin the serogroup.

What is claimed is:
 1. A method for the prevention of invasivepneumococcal disease (IPD) and pneumococcal pneumonia (PP) in patientscomprising administering a multivalent immunogenic composition to thepatient, wherein the multivalent immunogenic composition comprises S.pneumoniae polysaccharide carrier protein conjugates, wherein each ofthe conjugates comprises a polysaccharide of a particular S. pneumoniaeserotype conjugated to a carrier protein, wherein the S. pneumoniaeserotypes consist of 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F,31, 35B, 8, 9N, 10A, 11A, 12F, de-O-acetylated 15B, 17F, and 20A, andthe carrier protein is CRM197.
 2. The method of claim 1, wherein thede-O-acetylated 15B polysaccharide has an O-Acetyl content per repeatingunit of less than 5%.